Raf dimers and uses thereof

ABSTRACT

Disclosed herein are mutated RAF and KSR nucleic acids and polypeptides. Also disclosed are methods of using the mutated RAF and KSR to inhibit the dimerization of RAF/RAF and RAF/KSR. Also disclosed are methods of using the mutated RAF and KSR to screen for inhibitors of dimerization.

RELATED APPLICATIONS

This application is a continuation of International Application No.PCT/CA2010/001164, which designated the United States and was filed onJul. 23, 2010, published in English, which claims the benefit of U.S.Provisional Application No. 61/228,273, filed on Jul. 24, 2009. Theentire teachings of the above application(s) are incorporated herein byreference.

TECHNICAL FIELD

The present generally concerns mutated RAF and KSR isoforms, and moreparticularly to their inhibition of RAF/RAF and RAF/KSR dimer formation.

BACKGROUND

The ERK (extracellular signal-regulated kinase) pathway is anevolutionarily conserved signal transduction module that controlscellular growth, differentiation and survival (Wellbrock, C.,Karasarides, M. & Marais, R. The RAF proteins take centre stage, Nat RevMol Cell Biol., 5, 875-85 (2004)). Activation of receptor tyrosinekinases (RTKs) by the binding of growth factors initiates GTP loading ofRAS, which triggers the initial steps in the activation of the ERKpathway by modulating RAF family kinase function. Once activated, RAFparticipates in a sequential cascade of phosphorylation events thatactivate MEK, and in turn ERK. Unbridled signaling through the ERKpathway caused by activating mutations in RTKs, RAS or RAF, have beenlinked to a multitude of human cancers (Roberts, P. J. & Der, C. J.,Targeting the Raf-MEK-ERK mitogen-activated protein kinase cascade forthe treatment of cancer, Oncogene, 26, 3291-310 (2007)). Of note, onemember of the RAF family, B-RAF, is the most frequently mutated oncogenewithin the kinase superfamily (Greenman, C. et al., Patterns of somaticmutation in human cancer genomes, Nature, 446, 153-8 (2007)).

Not surprisingly, there has been a colossal effort to understand theunderlying regulation of this family of kinases. Despite intensescrutiny, the mechanisms governing RAF activation remain only partiallyunderstood. In particular, the process by which its kinase domainbecomes catalytically activated towards its substrate MEK remainselusive.

A greater understanding of the mechanisms that govern RAF activationwould be useful as a means to identify novel therapeutic interventionstrategies for disease such as cancer.

BRIEF SUMMARY

The following addresses the shortcomings of the above.

In one aspect, there is provided a composition comprising: an aqueoussolution of RAF/RAF homodimer. The composition includes equimolaramounts of RAF monomers. Each RAF monomer includes a RAF kinase domainhaving a dimerization interface. The RAF/RAF homodimer is a side-to-sidedimer having a 2 fold axis of symmetry.

In one aspect, there is provided a composition comprising: an aqueoussolution of RAF/KSR heterodimer. The composition includes equimolaramounts of KSR and RAF monomers. The KSR and the RAF monomer eachinclude a kinase domain having a dimerization interface. The heterodimerhas a 2-fold axis of symmetry.

In one aspect, there is provided a substantially pure nucleic acidencoding a mutated RAF polypeptide. The nucleic acid is DNA whichcontains the RAF gene. The DNA is genomic DNA or cDNA. The mutated RAFpolypeptide is mutated A-RAF, mutated B-RAF or mutated C-RAF. Themutated RAF polypeptide includes at least one mutated residue located ina dimerization interface. The mutated residue is selected from the groupconsisting of: H449, G450, R481H, L487, F488, M489, Y538, A541 and K542.The mutation is selected from the group consisting of: H449E, G450W,R481H, L487R, F488A, F488L, M489W, Y538F, A541E and K542E. The mutatedRAF polypeptide comprises a sequence of SEQ ID NO: 7, SEQ ID NO: 9, orSEQ ID NO: 15. The nucleic acid comprises a sequence of SEQ ID NO: 8,SEQ ID NO: 10 or SEQ ID NO: 16. The nucleic acid is DNA which isoperably linked to regulatory sequences for expression of a mutated RAFpolypeptide and wherein the regulatory sequences comprise a promoter.The promoter is a constitutive promoter, is inducible by one or moreexternal agents, or is cell-type specific.

In another aspect, there is provided a method of producing a mutated RAFpolypeptide, the method comprising:

-   -   a) providing a cell transfected with a nucleic acid sequence        encoding a mutated RAF polypeptide positioned for expression in        the cell;    -   b) culturing the transfected cells under conditions for        expressing the nucleic acid; and    -   c) producing the mutated RAF polypeptide.

In another aspect, there is provided a substantially pure mammalianmutated RAF polypeptide, or fragment thereof, the polypeptide beingencoded by the nucleic acid, as described above. The polypeptidecomprises an amino acid sequence substantially identical to an aminoacid sequence of SEQ ID NO: 7 or SEQ ID NO: 9. The polypeptide includesat least one mutant selected from the group consisting of: H449E, G450W,R481H, L487R, F488A, F488L, M489W, Y538F, A541E and K542E.

In one aspect, there is provided a substantially pure nucleic acidencoding a mutated KSR polypeptide. The nucleic acid is DNA whichcontains the KSR gene. The DNA is genomic DNA or cDNA. The mutated KSRpolypeptide is mutated KSR-1 or KSR-2. The mutated KSR polypeptideincludes at least one mutated residue located in a dimerizationinterface. The mutated residue is selected from the group consisting of:H699, G700, R732, L738, F739, M740, Y790, A793 and R794. The mutation isselected from the group consisitng of: H699E, G700W, R732H, L738R,F739A, F739L, M740W, Y790F, A793E and R794E. The mutated KSR polypeptidecomprises a sequence of SEQ ID NO: 11, SEQ ID NO: 13, or SEQ ID NO: 17.The nucleic acid comprises a sequence of SEQ ID NO: 12, SEQ ID NO: 14 orSEQ ID NO: 18. The nucleic acid is DNA which is operably linked toregulatory sequences for expression of the polypeptide and wherein theregulatory sequences comprise a promoter. The promoter is a constitutivepromoter, is inducible by one or more external agents, or is cell-typespecific.

In another aspect, there is provided a method of producing a mutated KSRpolypeptide, the method comprising:

-   -   a) providing a cell transfected with a nucleic acid sequence        encoding a mutated KSR polypeptide positioned for expression in        the cell;    -   b) culturing said transfected cells under conditions for        expressing the nucleic acid; and    -   c) producing the mutated KSR polypeptide.

In another aspect, there is provided a substantially pure mammalianmutated KSR polypeptide, or fragment thereof, the polypeptide beingencoded by the nucleic acid, as described above. The polypeptidecomprises an amino acid sequence substantially identical to an aminoacid sequence of SEQ ID NO: 11, SEQ ID NO: 13 or SEQ ID NO: 17. Thepolypeptide includes at least one mutant selected from the groupconsisting of: H699E, G700W, R732H, L738R, F739A, F739L, M740W, Y790F,A793E and R794E.

The polypeptide or the nucleic acid, as described above is mammalian.The mammal is murine or human.

The polypeptide or the nucleic acid, as described above, isnon-mammalian. The non-mammal is Drosophila melanogaster.

In another aspect, there is provided a vector comprising the nucleicacid, as described above, the vector being capable of directingexpression of the polypeptide encoded by the nucleic acid in avector-containing cell.

In another aspect, there is provided a cell that contains the nucleicacid, as described above.

In another aspect, there is provided a transgenic cell that contains thenucleic acid, as described above, wherein the nucleic acid is expressedin the transgenic cell.

In another aspect, there is provided a transgenic non-human mammalgenerated from the cell, as described above, wherein the nucleic acid isexpressed in the transgenic mammal. The transgenic non-human mammal is amouse.

The cell, as described above, is a mammalian cell, a yeast cell, or abacterial cell.

In one aspect, there is provided a method of detecting the presence of amutation in a RAF kinase domain, the method comprising:

-   -   a) providing a WT RAF kinase domain and a suspected mutant RAF        kinase domain, each domain having a cysteine residue located at        its N-terminus;    -   b) incubating the WT RAF kinase domain and the suspected mutant        RAF kinase domain with different cross-linking detectable        labels;    -   c) incubating together equimolar amounts of the labeled WT RAF        kinase domain and detecting a signal from the detectable label        so as to provide a dimerization reference signal; and    -   d) incubating equimolar amounts of the labeled suspected mutant        B-RAF kinase domain and detecting a signal from the detectable        labels, an absent signal or a reduce signal compared to that of        the dimerization reference signal being an indication that a        mutant B-RAF kinase domain is present.

In one aspect, there is provided a method of monitoring the formation ofRAF/RAF or RAF/KSR kinase domain dimers to detect mutations inhibitingdimerization or drug-like molecules interfering with dimerization, themethod comprising:

-   -   a) fusing either (i) a RAF kinase domain or (ii) a KSR kinase        domain at either of their N- or C-termini to a BRET donor or a        BRET acceptor to produce donor labeled and acceptor labeled        fusion proteins;    -   b) expressing the fusion proteins to identify combinations that        provide specific BRET signals;    -   c) introducing dimer interface mutations into either of the        labeled fusion proteins;    -   d) expressing the labeled mutated fusion proteins with WT RAF or        KSR kinase domains;    -   e) measuring the BRET signals, a loss or significant reduction        of the BRET signal using dimer interface mutations as opposed to        mutations remote from the interface, being an indication that a        specific BRET signal which depends on the RAF/RAF or RAF/KSR        dimerization interface has been obtained.

The BRET donor is renilla luciferase variant II or rlucII. The BRETacceptor is GFP10. The acceptor label is Yellow Fluorescent Protein(YFP). The donor labeled fusion protein comprises a sequence selectedfrom the group consisting of: SEQ ID NO: 24, SEQ ID NO: 34, SEQ ID NO:42 and SEQ ID NO: 48. The acceptor labeled fusion protein comprises asequence selected from the group consisting of: SEQ ID NO: 22, SEQ IDNO: 30, SEQ ID NO: 40 and SEQ ID NO: 54. The donor labeled mutatedfusion proteins comprise sequences SEQ ID NO: 36 and SEQ ID NO: 50. Theacceptor labeled mutated fusion proteins comprises a sequence of SEQ IDNO: 32.

In another aspect, there is provided a method of identifying a potentialinhibitor of RAF/RAF homodimerization, the method comprising.

-   -   a) fusing a RAF kinase domain at either of its N- or C-termini        to a BRET donor or a BRET acceptor to produce donor labeled and        acceptor labeled fusion proteins;    -   b) expressing the fusion proteins to identify combinations that        provide specific BRET signals;    -   c) introducing dimer interface mutations into either of the        labeled fusion proteins;    -   d) expressing the labeled mutated fusion proteins with WT RAF        kinase domains;    -   e) contacting the interface with the potential inhibitor; and    -   f) measuring the BRET signals, a loss or significant reduction        of the BRET signal for the wild-type RAF/RAF BRET pair being an        indication that the inhibitor is specifically bound to the        interface.

In another aspect, there is provided a method of identifying a potentialinhibitor of RAF/RAF homodimerization, the method comprising:

-   -   a) detectably labeling at least one of the dimerization        interface residues to generate a detectably labeled RAF monomer;    -   b) incubating the detectably labeled RAF monomer with the        potential inhibitor and a non-labeled RAF monomer;    -   c) measuring a signal from the detectable label;    -   d) contacting the RAF dimerization interface with the inhibitor        to determine the ability of the potential inhibitor to inhibit        RAF/RAF homodimerization.

The interface residues include H449, G450, R481, L487, F488, M489, Y538,A541 or K542.

In one aspect, there is provided a method of identifying a potentialinhibitor of RAF/RAF homodimerization, the method comprising.

-   -   a) fusing a RAF kinase domain at either of its N- or C-termini        to a BRET donor or a BRET acceptor to produce donor labeled and        acceptor labeled fusion proteins;    -   b) expressing the fusion proteins to identify combinations that        provide specific BRET signals;    -   c) introducing dimer interface mutations into either of the        labeled fusion proteins;    -   d) expressing the labeled mutated fusion proteins with WT RAF        kinase domains;    -   e) contacting the interface with the potential inhibitor; and    -   f) measuring the BRET signals, a loss or significant reduction        of the BRET signal for the wild-type RAF/RAF BRET pair being an        indication that the inhibitor is specifically bound to the        interface.

In another aspect, there is provided a method of identifying compoundsthat bind to a RAF or a KSR dimerization interface, the methodcomprising:

-   -   a) contacting the interface with a probe to form a probe:        interface complex, the probe being displaceable by a test        compound;    -   b) measuring a signal from the probe so as to establish a        reference level;    -   c) incubating the probe:interface complex with the test        compound;    -   d) measuring the signal from the probe;    -   e) comparing the signal from step d) with the reference level, a        modulation of the signal being an indication that the test        compound binds to the BIR domain, wherein the probe is a        compound labeled with a detectable label or an affinity label.

In another aspect, there is provided a method of identifying a potentialinhibitor of RAF/RAF homodimerization, the method comprising:

-   -   a) using the atomic coordinates of at least one of the interface        residues to generate a three dimensional structure of a RAF        dimerization interface;    -   b) using the three-dimensional structure to design or select the        potential inhibitor;    -   c) synthesizing the inhibitor; and    -   d) contacting the RAF dimierization interface with the inhibitor        to determine the ability of the potential inhibitor to inhibit        RAF/RAF homodimerization.

The interface residues are H449, G450, R481, L487, F488, M489, Y538,A541 or K542.

In one aspect, there is provided a method of identifying a potentialinhibitor of RAF/KSR heterodimerization, the method comprising:

-   -   a) using the atomic coordinates of at least one of interface        residues to generate a three dimensional structure of a KSR        dimerization interface;    -   b) using the three-dimensional structure to design or select the        potential inhibitor;    -   c) synthesizing the inhibitor; and    -   d) contacting the KSR dimerization interface with the inhibitor        to determine the ability of the potential inhibitor to inhibit        RAF/KSR heterodimerization.

The interface residues are H699, G700, R732, L738, F739, M740, Y790,A793 or R794.

In another aspect, there is provided a method of detecting in a subjectthe susceptibility to develop a condition or an increased likelihood ofdeveloping a condition characterized by impaired regulation of proteinRAF or KSR dimerization, the method comprising:

-   -   a) obtaining from said subject a biological sample having DNA;    -   b) sequencing predetermined regions of said DNA encoding a RAF        or KSR polypeptide; and    -   c) comparing the sequence obtained at (b) with a corresponding        sequence from a non-susceptible control subject for identifying        a RAF or KSR mutation known to be indicative of the        susceptibility.

In another aspect, there is provided a human RAF or KSR polypeptidewhich comprises a mutation compared to wild type RAF or KSR, whereinsaid mutation produces a mutant version of human RAF or KSR polypeptidethat includes at least one mutant H449, G450, R481, L487, F488, M489,Y538, A541 or K542 residue, and wherein the mutant version prevents theformation of a RAF/RAF homodimer.

In another aspect, there is provided a human RAF kinase domain whichcomprises a mutated dimerization interface having at least one mutantH449, G450, R481H, L487, F488, M489, Y538, A541 or K542 residue.

In another aspect, there is provided a human KSR kinase domain whichcomprises a mutated dimerization interface having at least one mutantH699, G700, R732, L738, F739, M740, Y790, A793 or R794 residue.

In another aspect, there is provided a substantially pure nucleic havingthe sequence of full length RAF and encoding the polypeptide sequencesof SEQ ID NO: 7 and SEQ ID NO: 9.

In another aspect, there is provided a substantially pure nucleic acidhaving about 50% or greater nucleotide sequence identity to thesequences, as described above.

In another aspect, there is provided a substantially pure nucleic havingthe sequence of full length KSR and encoding the polypeptide sequencesof SEQ ID NO: 11, SEQ ID NO: 13 and SEQ ID NO: 17.

In another aspect, there is provided a substantially pure nucleic acidhaving about 50% or greater nucleotide sequence identity to thesequences of SEQ ID NO: 12, SEQ ID NO: 14 and SEQ ID NO: 18.

In another aspect, there is provided a cell in vitro expressing arecombinant nucleic acid comprising a nucleic acid sequence encoding amutated RAF polypeptide, as described above. In one example, the cell isa mammalian cell, a yeast cell, or a bacterial cell.

In one aspect, there is provided a method of producing a drug whichinhibits RAF/RAF homodimerization, the method comprising: identifying adrug or designing a drug which interacts with at least one of the H449,G450, R481, L487, F488, M489, Y538, A541 and K542 residues; andsynthesizing the drug.

In another aspect, there is provided a method of producing a drug whichinhibits RAF/KSR heterodimerization, the method comprising: identifyinga drug or designing a drug which interacts with at least one of theH699, G700, R732, L738, F739, M740, Y790, A793 and R794 residues; andsynthesizing the drug.

In another aspect, there is provided a composition comprising: aninhibitor adapted to inhibit the formation of a RAF/RAF homodimer or aRAF/KSR heterodimer, in which the inhibitor binds to at least one of theH449, G450, R481, L487, F488, M489, Y538, A541 and K542 residues in aRAF monomer or at least one of the H699, G700, R732, L738, F739, M740,Y790, A793 and R794 residues in a KSR monomer.

In another aspect, there is provided a method of treating or preventinga disease in a subject, the disease being characterized by RAF/RAFhomodimerization or RAF/KSR heterodimerization, the method comprising:administering to the subject in need thereof, an expression vectorencoding mutated RAF or KSR polypeptide, the mutated RAF or KSRpolypeptide being positioned in the vector for expression in a cell ofthe subject in which RAF/RAF homodimerization or RAF/KSRheterodimerization is taking place, so as to treat or prevent thedisease.

In another aspect, there is provided a dominant negative mutantpolypeptide of mammalian RAF or KSR, wherein the mutant polypeptidecomprises a kinase domain having a dimerization interface and does notbind to a WT mammalian RAF or KSR dimerization interface.

In another aspect, there is provided a purified antibody whichspecifically binds to a mammalian mutated RAF or KSR polypeptide. Themammal is a human. The mammal is a mouse. The mutated RAF polypeptide isB-RAF. The KSR polypeptide is KSR-1. The antibody is a polyclonalantibody. The antibody is a monoclonal antibody.

BRIEF DESCRIPTION OF THE FIGURES

In order that the herein described may be readily understood, certainembodiments are illustrated by way of example in the accompanyingdrawings.

FIG. 1—KSR possesses intrinsic RAF activating potential. A)Co-overexpression of KSR, RAF and its substrate MEK as indicated in S2cells leads to activation of RAF in a KSR concentration-dependent mannerin the presence or absence of RNAi-mediated knockdown of RAS orco-overexpression of a constitutively active RAS^(V12) variant. RAFactivation was assessed by immunoblotting for phospho-MEK. Thecatalytically-inactive RAF K455M (KM) mutant served as a negativecontrol.B and C) The RAF activation potential of overexpressed KSR isnot affected by RNAi-mediated knockdown of CNK, HYP or CK2α or bymutation of the proposed CK2 α regulatory sites in KSR (T399A/K402A) andRAF (S416A/S417A). Assessment of the RNAi-mediated knock-downs forendogenous RAS, CNK, HYP and CK2 α is provided in FIG. 13.

FIG. 2—KSR_R732H mutation abolishes its inherent RAF activatingpotential. Wild type KSR but not the KSR_R732H mutant is able to driveRAF activation in an S2 cell overexpression system. Experiments wereperformed as in FIG. 1.

FIG. 3—A side-to-side dimer configuration of RAF underlies an allostericmode of regulation. A) Projection of highly conserved residues acrossboth KSR and RAF orthologues onto the crystal structure (PDB ID=1UWH)(Wan, P. T. et al., Mechanism of activation of the RAF-ERK signalingpathway by oncogenic mutations of B-RAF, Cell, 116, 855-67 (2004)) ofthe B-RAF kinase domain (top panel) highlights common side-to-side dimercontact surfaces visualized originally in crystal structures of B-RAF(bottom panel). B) Crystal structure of B-RAF highlighting the positionof Arg481 (equivalent to Arg732 in KSR) at the center of theside-to-side dimer interface (PDB ID=1UWH) (Wan, P. T. et al., Mechanismof activation of the RAF-ERK signaling pathway by oncogenic mutations ofB-RAF, Cell, 116, 855-67 (2004)). Residue numbering scheme correspondsto Drosophila RAF. One protomer is displayed as a surface representationin orange and the other is shown as a ribbons representation in violet.Inset displays a close-up view of hydrogen bonding interactionsinvolving Arg481, an ordered solute molecule, and main-chain carbonylgroups in the linker joining helix α C to strand β4.C) Analyticalultracentrifugation analysis reveals that mutation of Arg481 (R481H) inB-RAF transitions the protein from a dimer (left panel) to a monomer(right panel) in solution. The red line denotes a fitted curve to theself-association model. The residuals for the fit are shown in the upperpanels.

FIG. 4—Side-to-side dimer interface residues are conserved in all KSRand RAF proteins. Sequence alignment of the kinase domains of KSR andRAF from divergent organisms highlighting conserved residues. Forcomparison, the sequence of the kinase domains of LCK and PKA areco-aligned demonstrating that the side-to-side dimer contact residuesare unique to the KSR/RAF family. The sequence of the kinase domainN-lobe is boxed in red and the secondary structural elements areindicated above the sequence. Aligned sequences correspond to those fromDrosophila (d), human (h), mouse (m), zebrafish (z), and chicken (c).Only the B-RAF sequence is shown for species where multiple RAF isoformsexist.

FIG. 5—Perturbing the side-to-side dimer interface on RAF and KSRimpairs RAF activation. A) Left panel: Model of a side-to-sideheterodimer between KSR and RAF kinase domains. RAF is displayed as asurface representation in purple while KSR is shown in ribbonsrepresentations in green. Highlighted in red stick representation arethe positions of residues selected for mutational analysis in KSR(G700W, R732H, F739A, M740W and Y790F). Position of analogous mutatedsites in RAF (G450W, R481H, F488A, M489W and Y538F, respectively) aredenoted by yellow surface; residue numbering scheme corresponds toDrosophila RAF. Right panel: The individual effect of KSR and RAFmutations on RAF activation was assessed by monitoring the levels ofphosphorylated MEK in S2 cells as performed in FIG. 1. Control mutationsoutside the side-to-side dimer interface correspond to K460A, E601A andM640A in RAF, and D710A, E859A and V898A in KSR. B) Left panel:Schematic for induced side-to-side dimer formation using FRB/FKBPfusions to the kinase domains of KSR and RAF. Right panel: The RAFactivation potential of the FRB/FKBP fused kinase domains of KSR and RAFwere assessed by monitoring the levels of phosphorylated MEK in thepresence or absence of rapamycin in S2 cells as performed in FIG. 1. C)Left panel: Schematic for induced side-to-side homodimer formation ofRAF kinase domains. The catalytically-inactive (K455S) FRB-RAF fusion isindicated by an ‘X’ within the N-lobe. Right panel: Activation potentialof FRB/FKBP RAF homodimers was assessed as in FIG. 3B.

FIG. 6—The kinase domain of RAF adopts a side-to-side dimericconfiguration in the crystal structure. A) The side-to-side dimerconfiguration of the kinase domain of B-RAF is shown viewedperpendicular to the 2-fold axis of symmetry (PDB ID=1UWH) (Wellbrock,C., Karasarides, M. & Marais, R., The RAF proteins take centre stage,Nat Rev Mol Cell Biol, 5, 875-85 (2004)). The N-lobes of the two kinasedomains, which compose the majority of the dimer interaction surfaces,are colored in darker tint. B) Superposition of the six reported B-RAFkinase domain structures reveal an identical mode of side-to-sidedimerization (PDB IDs: 1UWH (Wan, P. T. et al., Mechanism of activationof the RAF-ERK signaling pathway by oncogenic mutations of B-RAF, Cell,116, 855-67 (2004)), 1UWJ (Wan, P. T. et al., Mechanism of activation ofthe RAF-ERK signaling pathway by oncogenic mutations of B-RAF, Cell,116, 855-67 (2004)), 2FB8 (King, A. J. et al., Demonstration of agenetic therapeutic index for tumors expressing oncogenic BRAF by thekinase inhibitor SB-590885, Cancer Res, 66, 11100-5 (2006)), 3C4C (Tsai,J. et al., Discovery of a selective inhibitor of oncogenic B-Raf kinasewith potent antimelanoma activity, Proc Natl Acad Sci, USA, 105, 3041-6(2008)), 3C4D (Tsai, J. et al., Discovery of a selective inhibitor ofoncogenic B-Raf kinase with potent antimelanoma activity. Proc Natl AcadSci., USA, 105, 3041-6 (2008)) and 3D4Q (Hansen, J. D. et al., Potentand selective pyrazole-based inhibitors of B-Raf kinase, Bioorg Med ChemLett, 18, 4692-5 (2008)). C) Comparison of the B-RAF side-to-side modeof dimerization with the specific mode of dimerization of PKR (PDBID=2A19 (Dar, A. C., Dever, T. E. & Sicheri, F., Higher-order substraterecognition of eIF2alpha by the RNA-dependent protein kinase PKR, Cell,122, 887-900 (2005)) and EGFR (PDB ID=2GS2 (Zhang, X., Gureasko, J.,Shen, K., Cole, P. A. & Kuriyan, J., An allosteric mechanism foractivation of the kinase domain of epidermal growth factor receptor,Cell, 125, 1137-49 (2006)) kinase domains. Helix αC is a participant inall three modes of dimerization.

FIG. 7—An engineered KSR/RAF chimera can functionally mimic wildtypeKSR. A) Schematics of wild type and chimeric KSR/RAF constructsinvolving either a full RAF kinase domain swap into KSR (Chimera-A) orjust a RAF N-lobe swap into KSR (Chimera-B). B) The ability of wild typeRAF, KSR, and KSR/RAF chimeric constructs to drive RAF activation in S2cells was assessed by the levels of phosphorylated MEK.

FIG. 8—Perturbing the side-to-side dimer interface on RAF and KSRimpairs RAF activation. A) Model of a side-to-side heterodimer betweenKSR and RAF kinase domains. RAF is displayed as a surface representationin purple while KSR is shown in ribbons representations in green.Highlighted in red stick representation are the positions of residuesselected for mutational analysis in KSR (H699E, L738R, F739L, A793E, andR794E). Position of analogous mutated sites in RAF (H449E, L487R, F488L,A541E and K542E, respectively) are denoted by yellow surface; residuenumbering scheme corresponds to Drosophila RAF. B and C) The RAFactivation potential of the FRB/FKBP fused kinase domains of KSR and RAFwere assessed by monitoring the levels of phosphorylated MEK in thepresence or absence of rapamycin in S2 cells as illustrated in theschematic.

FIG. 9—KSR contains a putative 14-3-3 binding site C-terminal to itskinase domain. A) Sequence alignment of the C-terminus of KSR reveals ahighly conserved 14-3-3 recognition site common to that found in RAF(Drosophila RAF residue numbering is indicated above the alignment).Aligned sequences correspond to those from Drosophila (d), human (h),mouse (m), zebrafish (z), and chicken (c). B) S2 cell overexpressionassay for RAF activation showing the effects of RNAi-mediated knockdownof 14-3-3 isoforms or mutation of putative 14-3-3 binding sites in KSR(R950A/S951A) and RAF (S701A). The effect of RNAi on endogenous 14-3-3protein levels is shown in FIG. 13.

FIG. 10—Binding of 14-3-3 to KSR and RAF may promote the formation ofhetero- and/or homotypic dimers by the kinase domain. Structural modelshowing that the geometry of the KSR/RAF (or RAF/RAF) side-to-side dimeris compatible with the spatial requirements for binding to dimeric14-3-3 proteins. Surface representation of 14-3-3 bound tophospho-peptides is based on PDB ID 1YWT (Wilker, E. W., Grant, R. A.,Artim, S. C. & Yaffe, M. B., A structural basis for 14-3-3sigmafunctional specificity, J Biol Chem, 280, 18891-8 (2005)).

FIG. 11—Side-to-side dimer formation underlies the aberrant signalingpotential of oncogenic RAF mutants. A) RAF activation assay usingoverexpressed full-length RAF and MEK proteins in S2 cells. The dimerinterface mutation (RAF_R481H) abrogates the pronounced activationpotential of the activation segment mutation (analogous to oncogenicB-RAF mutation) RAF_T571E/T574D (denoted RAF-AL^(ED)). B) Glu558 locatesto the side-to-side dimer interface in RAF and is mutated to Lys inhuman cancers (E558K mutation; residue numbering scheme corresponds toDrosophila RAF). The longer Lys residue could potentially engage inhydrogen bonding interactions with Ser561 on the opposite protomer. C)Left panel: Schematic for induced side-to-side homodimer formation ofRAF kinase domains as in FIG. 3C. Right panel: Catalytically inactiveFRB-RAF harboring the E558K mutation was assessed for its activationpotential towards FKBP-RAF in trans as in FIG. 3C.

FIG. 12—Oncogenic B-RAF E558K mutation promotes kinase domaindimerization. Analytical ultracentrifugation analysis reveals that theoncogenic E558K mutation in B-RAF transitions the B-RAF_L487R dimermutant from weak monomer-dimer equilibrium (left panel) to a dimer(right panel) in solution; residue numbering scheme corresponds toDrosophila RAF. The red line denotes a fitted curve to theself-association model. The residuals for the fit are shown in the upperpanels.

FIG. 13—Depletion of specific endogenous targets by RNAi. To ensure thatdsRNAs directed against RAS, CNK, HYP, CK2α, 14-3-3ε or 14-3-3ζ workedas expected, we separately incubated S2 cells with specific dsRNAs (15μg/ml) against these intended targets and monitored their respectiveprotein or mRNA levels using either specific antibodies or qPCR. GFPdsRNA was used as negative control. In panel A, the effect of RASdepletion was also monitored by assessing phospho-MAPK (pMAPK) levelsinduced by the activated Sevenless (SEV^(S11)) RTK expressed under thecontrol of the hsp70 promoter (Laberge, G., Douziech, M., & Therrien, M.Src42 binding activity regulates Drosophila RAF by a novel CNK-dependentderepression mechanism, EMBO J, 24, 487-98 (2005)).

FIG. 14A shows polypeptide and polynucleotide sequences (SEQ ID NO: 7and 8) of homo sapiens v-raf murine sarcoma viral oncogene homolog B1(BRAF) showing mutated residues as underlined and highlighted.

FIG. 14B shows polypeptide and polynucleotide sequences (SEQ ID NO: 9and 10) of mus musculus Braf transforming gene (Braf) showing mutatedresidues as underlined and highlighted.

FIG. 14C shows polypeptide and polynucleotide sequences (SEQ ID NO: 11and 12) of homo sapiens kinase suppressor of ras 1 (KSR 1) showingmutated residues as underlined and highlighted.

FIG. 14D shows polypeptide and polynucleotide sequences (SEQ ID NO: 13and 14) of mus musculus kinase suppressor of ras 1 (Ksr 1) showingmutated residues as underlined and highlighted.

FIG. 14E shows polypeptide and polynucleotide sequences (SEQ ID NO: 15and 16) of Drosophila melanogaster pole hole (phi) transcript variant Ashowing mutated residues as underlined and highlighted.

FIG. 14F shows polypeptide and polynucleotide sequences (SEQ ID NO: 17and 18) of Drosophila melanogaster kinase suppressor of ras (ksr)showing mutated residues as underlined and highlighted.

FIG. 15—Development of a Bioluminescence Resonance Energy Transfer(BRET) assay to monitor RAF/RAF homodimerization. (A) Structure of thehuman BRAF kinase. RBD, CRD and Ser/Thr stand for Ras-Binding Domain,Cysteine-Rich Domain and Ser/Thr-rich domains respectively. The Kinasedomain (KD) and its C-terminal extension (dashed box) were used in allBRET constructs described here. (B) Structure of the BRAF-KD donor(rlucII) and acceptor (GFP10) expression constructs used in the BRETassay. (C) Saturation curve of the BRAF-KD-wt and BRAF-KD-R481H allelesshowing a significant reduction in the BRET_(max) and BRET₅₀ when adimer interface mutation (R481H) is introduced in the BRAF-KD. (D)Parameters derived from fit of our data with a hyperbolic function. R²denotes the goodness of fit of our data to a hyperbolic function.BRET_(max) and BRET₅₀ were interpolated using the hyperbolic function.

FIG. 16 shows CAAX-box and BRET donor and acceptor polypeptide sequences(human KRAS CAAX-box CDS: SEQ ID NO: 19; human KRAS CAAX-box: SEQ ID NO:20; GFP10 CDS: SEQ ID NO: 21; GFP10: SEQ ID NO: 22; rlucII CDS: SEQ IDNO: 23; and rlucII: SEQ ID NO: 24).

FIG. 17 shows human BRAF (hBRAF) polypeptide sequences (hBRAF-KD-wt CDS:SEQ ID NO: 25; hBRAF-KD-wt: SEQ ID NO: 26; hBRAF-KD-R481H CDS: SEQ IDNO: 27; hBRAF-KD-R481H: SEQ ID NO: 28). The bolded residues indicatelinker and restriction sites. Mutated residues are shaded in black.

FIG. 18 shows human BRAF-KD clones between the NheI and XbaI sites inpCDNA3.1-zeo (GFP10-hBRAF-KD-wt-CAAX CDS: SEQ ID NO: 29;GFP10-hBRAF-KD-wt-CAAX: SEQ ID NO:30; GFP10-hBRAF-KD-R481H-CAAX CDS: SEQID NO: 31; GFP10-hBRAF-KD-R481H-CAAX: SEQ ID NO: 32;rlucII-hBRAF-KD-wt-CAAX CDS: SEQ ID NO: 33: rlucII-hBRAF-KD-wt CAAX: SEQID NO: 34; rlucII-hBRAF-KD-R481H-CAAX CDS: SEQ ID NO: 35; andrlucII-hBRAF-KD-R481H-CAAX: SEQ ID NO: 36). The bolded residues indicatelinker and restriction sites. Mutated residues are shaded in black.

FIG. 19 shows human CRAF (hCRAF) polypeptide sequences (hCRAF-KD-wt CDS:SEQ ID NO: 37: hCRAF-KD-wt: SEQ ID NO: 38).

FIG. 20 shows hCRAF-KD fusions that are cloned between NheI and XbaI inpCDNA3.1-zeo (GFP10-hCRAF-KD-wt-CAAX CDA: SEQ ID NO: 39;GFP10-hCRAF-KD-wt-CAAX: SEQ ID NO: 40; rlucII-hCRAF-KD-wt-CAAX CDS: SEQID NO: 41; and rlucII-hCRAF-KD-wt-CAAX: SEQ ID NO: 42). The boldedresidues indicate linker and restriction sites.

FIG. 21 shows human KSR1 (hKSR1) sequences (hKSR1-KD-wt-CDS: SEQ ID NO:43; hKSR1-KD-wt: SEQ ID NO: 44; hKSR1-KD-C922Y CDS: SEQ ID NO: 45; andhKSR1-KD-C922Y: SEQ ID NO: 46). The bolded residues indicate linker andrestriction sites. Mutated residues are shaded in black.

FIG. 22 shows human KSR1-KD-rlucII fusions cloned between KpnI and PmeIin pCDNA3.1-zeo (hKSR1-KD-wt-rlucII CDS: SEQ ID NO: 47;hKSR1-KD-wt-rlucII: SEQ ID NO: 48; hKSR1-KD-C922Y-rlucII CDS: SEQ ID NO:49; and hKSR1-KD-C922Y-rlucII: SEQ ID NO: 50). The bolded residuesindicate linker and restriction sites.

FIG. 23 shows human MEK1 (hMEK1) sequences (hMEK1 CDS: SEQ ID NO: 51;and hMEK1: SEQ ID NO: 52).

FIG. 24 shows human GFP10-MEK1 full length fusion cloned between NheIand XbaI in pCDNA3.1-zeo (GFP10-hMEK1 CDS: SEQ ID NO: 53; andGFP10-hMEK1: SEQ ID NO: 54). The bolded residues indicate linker andrestriction sites.

FIG. 25 shows the sequences of cloning oligonucleotides. Human BRAF(OL5_hBRAF_KD_+start_F: SEQ ID NO: 55; OL3_hBRAF_KD_+CAAX_+stop_R: SEQID NO: 56); human CRAF (OL5_hCRAF_KD_+start_F: SEQ ID NO: 57;OL5_hCRAF_KD_+CAAX_+stop_R: SEQ ID NO: 58); human KSR1(OL5_hKSR1_KpnI_cloning_BRET: SEQ ID NO: 59; OL3_hKSR_XbaI_cloning_BRET:SEQ ID NO:60); and human MEK (OL5_hMEK1_KpnI_cloning_BRET: SEQ ID NO:61; OL3_hMEK_XbaI_cloning_BRET: SEQ ID NO: 62). The bolded residuesindicate linker and restriction sites.

FIG. 26 shows sequencing oligonucleotides (OL5_hBRAF_seq1_F: SEQ ID NO:63; OL5_hBRAF_seq2_F: SEQ ID NO: 64; OL5_hCRAF_seq1_F: SEQ ID NO: 65;and OL5_hCRAF_seq2_F: SEQ ID NO: 66).

FIG. 27 shows mutagenesis oligonucleotides. The following primer pairwere used to generate the side-to-side dimer interface mutant R481H inBRAF: OL5_hBRAF_R481H_F (SEQ ID NO: 67); and OL3_hBRAF_R481H_R (SEQ IDNO: 68). The following primer pair was used to introduce the C922Y hMEK1interaction mutant in hKSR1: OL5_hKSR1_C922Y_F (SEQ ID NO: 69) andOL3_hKSR1_C922Y_R (SEQ ID NO: 70). The bolded residues indicate linkerand restriction sites.

DETAILED DESCRIPTION

In the following description of the embodiments, references to theaccompanying Figures are by way of illustration of an example by whichthe embodiments described herein may be practiced. It will be understoodthat other embodiments may be made without departing from the scope ofthat disclosed herein.

Definitions:

Unless otherwise specified, the following definitions apply throughout:

As used herein, the singular forms “a”, “an”, and “the” include pluralreferents unless the context clearly indicates otherwise. Thus, forexample, reference to “a mutation” includes one or more of suchmutations and reference to “the method” includes reference to equivalentsteps and methods known to those of ordinary skill in the art that couldbe modified or substituted for the methods described herein.

As used herein, the term “comprising” is intended to mean that the listof elements following the word “comprising” are required or mandatorybut that other elements are optional and may or may not be present.

As used herein, the term “consisting of” is intended to mean includingand limited to whatever follows the phrase “consisting of”. Thus thephrase “consisting of” indicates that the listed elements are requiredor mandatory and that no other elements may be present.

As used herein, the term “RAF” is intended to refer to a protein, apolypeptide or fragment thereof, encoded by a RAF gene. Examples ofWild-type (WT) human RAF proteins include the RAF protein isoforms knownas A-RAF, B-RAF and C-RAF (e.g., genbank accession numbers P10398 forHomo sapiens A-RAF; P15056 for Homo sapiens B-RAF; and PO4049 for Homosapiens C-RAF). Examples of RAF xenologues are (e.g. genbank accessionnumber P11346 for Drosophila melanogaster pole hole (phl; RAF); P04627for Mus musculus A-RAF; P28028 for Mus musculus B-RAF; and Q99N57 forMus musculus C-RAF. Included in this definition are any functional RAFfragment, or any fusion of functional RAF fragments. Examples of thesefragments include those that consist of, consist essentially of, orcomprise the RAF kinase domain. Furthermore, the term also encompassesany fusion of full length RAF, or a functional fragment thereof, withanother polypeptide. These fusions include, but are not limited to,GST-RAF, HA tagged RAF, or Flag tagged RAF. These additionalpolypeptides may be linked to the N-terminus and/or C-terminus of RAF.Chimeric RAF protein, including a protein comprising a fusion of a RAFdomain or domains with a portion of another protein, wherein thechimeric RAF retains the properties of human RAF, are also included.Examples of chimeric RAF proteins include the fusion of any of the aboveRAF domains, or fragments thereof, to any domain or fragment of thefollowing proteins such as, for example, GST, luciferase or GFPderivatives. RAF also includes any protein with at least 70% sequenceidentity with mammalian or non-mammalian RAF. The term also includes anyconservative substitutions of amino-acid residues in RAF. The term“conservative substitution” refers to replacement of an amino acidresidue by a chemically similar residue, e.g., a hydrophobic residue fora separate hydrophobic residue, a charged residue for a separate chargedresidue, etc. Examples of conserved substitutions for non-polar R groupsare alanine, valine, leucine, isoleucine, proline, methionine,phenylalanine, and tryptophan. Examples of substitutions for polar, butuncharged R groups are glycine, serine, threonine, cysteine, asparagine,or glutamine. Examples of substitutions for negatively charged R groupsare aspartic acid or glutamic acid. Examples of substitutions forpositively charged R groups are lysine, arginine, or histidine.Furthermore, the term RAF includes conservative substitutions withnon-natural amino-acids.

The following are Accession numbers for RAF cDNA and protein sequencesfor various species:

Accession numbers for RAF cDNA sequences

NM_(—)080308: Drosophila melanogaster pole hole (phl; RAF)

NM_(—)009703: Mus musculus A-RAF

NM_(—)139294: Mus musculus B-RAF

AB057663: Mus musculus C-RAF

X04790: Homo sapiens A-RAF

NM_(—)004333: Homo sapiens B-RAF

NM_(—)002880: Homo sapiens C-RAF

Accession numbers for RAF protein sequences

P11346: Drosophila melanogaster pole hole (phl; RAF)

P04627: Mus musculus A-RAF

P28028: Mus musculus B-RAF

Q99N57: Mus musculus C-RAF

P10398: Homo sapiens A-RAF

P15056: Homo sapiens B-RAF

P04049: Homo sapiens C-RAF

As used herein, the terms “mutated RAF protein” and “mutated RAFpolypeptide” are used interchangeably throughout and are intended tomean a WT RAF protein in which one or more amino acid residues have beenchanged. In certain examples described herein, the mutations includeH449E, G450W, R481H, L487R, F488A, F488L, M489W, Y538F, A541E and K542E,which are located in the dimerization interface. Unless otherwisestated, amino acid residue positions in RAF proteins refer to those ofthe Drosophila. melanogaster sequences.

As used herein, the term “KSR” is intended to refer to a KinaseSuppressor of Ras protein, a polypeptide or fragment thereof, encoded bya KSR gene. Examples of Wild type (WT) human KSR proteins include theKSR protein isoforms known as KSR1 and KSR2 (e.g. genbank accessionnumber A8MY87 for Homo sapiens kinase suppressor of ras 1 (KSR1) andQ6VAB6: for Homo sapiens kinase suppressor of ras 2 (KSR2). Examples ofKSR xenologues are (e.g. genbank accession numbers Q24171 for Drosophilamelanogaster kinase suppressor of ras (KSR); Q61097 for Mus musculuskinase suppressor of ras 1 (KSR1); and Q3UVC0 for Mus musculus kinasesuppressor of ras 2 (KSR2). The term “KSR” also means any functional KSRfragment, or any fusion of functional KSR fragments. Examples of thesefragments include those that consist of, consist essentially of, orcomprise the KSR kinase domain. Included in this definition are fusionof full length KSR, or a functional fragment thereof, with anotherpolypeptide. These fusions include, but are not limited to, GST-KSR, HAtagged KSR, or Flag tagged KSR. These additional polypeptides may belinked to the N-terminus and/or C-terminus of KSR. Any chimeric KSRprotein including a protein comprising a fusion of a KSR domain ordomains with a portion of another protein, wherein the chimeric KSRretains the properties of human KSR, are also included. Examples ofchimeric KSR proteins include the fusion of any of the above KSRdomains, or fragments thereof, to any domain or fragment of thefollowing proteins such as, for example, GST, luciferase or GFPderivatives. KSR also includes any protein with at least 70% sequenceidentity with mammalian or non-mammalian KSR. The term also includes anyconservative substitutions of amino-acid residues in KSR. The term“conservative substitution” refers to replacement of an amino acidresidue by a chemically similar residue, e.g., a hydrophobic residue fora separate hydrophobic residue, a charged residue for a separate chargedresidue, etc. Examples of conserved substitutions for non-polar R groupsare alanine, valine, leucine, isoleucine, proline, methionine,phenylalanine, and tryptophan. Examples of substitutions for polar, butuncharged R groups are glycine, serine, threonine, cysteine, asparagine,or glutamine. Examples of substitutions for negatively charged R groupsare aspartic acid or glutamic acid. Examples of substitutions forpositively charged R groups are lysine, arginine, or histidine.Furthermore, the term KSR includes conservative substitutions withnon-natural amino-acids.

The following are Accession numbers for KSR cDNA and protein sequencesfor various species:

Accession numbers for KSR cDNA sequences

NM_(—)079512: Drosophila melanogaster kinase suppressor of ras (KSR)

NM_(—)013571: Mus musculus kinase suppressor of ras 1 (KSR1)

DQ531035: Mus musculus kinase suppressor of ras 2 (KSR2)

NM_(—)014238: Homo sapiens kinase suppressor of ras 1 (KSR1)

NM_(—)173598: Homo sapiens kinase suppressor of ras 2 (KSR2)

Accession numbers for KSR protein sequences

Q24171: Drosophila melanogaster kinase suppressor of ras (KSR)

Q61097: Mus musculus kinase suppressor of ras 1 (KSR1)

Q3UVC0: Mus musculus kinase suppressor of ras 2 (KSR2)

Q8IVT5: Homo sapiens kinase suppressor of ras 1 (KSR1)

Q6VAB6: Homo sapiens kinase suppressor of ras 2 (KSR2)

As used herein, the terms “mutated KSR protein” and “mutated KSRpolypeptide” are used interchangeably throughout and are intended tomean a WT KSR protein in which one or more amino acid residues have beenchanged. In certain examples described herein, the mutations includeH699E, G700W, R732H, L738R, F739A, F739L, M740W, Y790F, A793E and R794E,

which are located in the dimerization interface. Unless otherwisestated, amino acid residue positions in KSR proteins refer to those ofthe Drosophila. melanogaster sequences.

As used herein, the term “mutation” is intended to mean any alterationin a gene which alters function or expression of the gene products, suchas mRNA and the encoded for protein. This includes, but is not limitedto, altering mutation, point mutation, truncation mutation, deletionmutation, frameshift mutation, and null mutation.

As used herein, the term “RAF gene” is intended to mean a gene encodinga RAF polypeptide having a dimerization interface. The RAF gene is agene having about 50% or greater nucleotide sequence identity to atleast one of human RAF isoforms (e.g. genbank accession numbers X04790for Homo sapiens A-RAF; NM_(—)004333 for Homo sapiens B-RAF; andNM_(—)002880 for Homo sapiens C-RAF. Examples of RAF xenologues are(e.g. genbank accession numbers NM_(—)080308 for Drosophila melanogasterpole hole (phl; RAF); NM_(—)009703 for Mus musculus A-RAF; NM_(—)139294for Mus musculus B-RAF; and AB057663 for Mus musculus C-RAF).

As used herein, the term “KSR gene” is intended to mean a gene encodinga KSR polypeptide having a dimerization interface. The KSR gene is agene having about 50% or greater nucleotide sequence identity to atleast one of human KSR isoforms (e.g. genbank accession numbersNM_(—)014238 for Homo sapiens kinase suppressor of ras 1 (KSR1); andNM_(—)173598 for Homo sapiens kinase suppressor of ras 2 (KSR2)).Examples of KSR xenologues are (e.g. genbank accession numbersNM_(—)079512 for Drosophila melanogaster kinase suppressor of ras (KSR);NM_(—)013571 for Mus musculus kinase suppressor of ras 1 (KSR1); andDQ531035 for Mus musculus kinase suppressor of ras 2 (KSR2).

As used herein, the term “gene” refers to a nucleic acid comprising anopen reading frame encoding a polypeptide, including both exon and(optionally) intron sequences. The nucleic acid may also optionallyinclude non-coding sequences such as promoter or enhancer sequences. Theterm “intron” refers to a DNA sequence present in a given gene that isnot translated into protein and is generally found between exons.

As used herein, the term “dimer interface” is intended to mean a site inthe WT RAF or KSR polypeptide sequence or the mutated RAF or KSRpolypeptide sequence, which reacts with a RAF or KSR substrate.

As used herein, the terms “RAF kinase domain” or “KSR kinase domain” areintended to mean the portion of the RAF or KSR proteins that are relatedin sequence to a generic protein kinase domain.

As used herein, the term “detectable label” is intended to mean acompound that may be linked to a RAF or KSR kinase domain, such thatwhen the compound is associated with the domain, the label allows eitherdirect or indirect recognition of the compound so that it may bedetected, measured and quantified.

As used herein, the term “affinity tag” is intended to mean a ligand orgroup, which is linked to a RAF or KSR kinase domain to allow anothercompound to be extracted from a solution to which the ligand or group isattached.

As used herein, the term “nucleic acid” or a “nucleic acid molecule” asused herein refers to any DNA or RNA molecule, either single or doublestranded and, if single stranded, the molecule of its complementarysequence in either linear or circular form. In discussing nucleic acidmolecules, a sequence or structure of a particular nucleic acid moleculemay be described herein according to the normal convention of providingthe sequence in the 5′ to 3′ direction. With reference to nucleic acidsdescribed herein, the term “isolated nucleic acid” is sometimes used.This term, when applied to DNA, refers to a DNA molecule that isseparated from sequences with which it is immediately contiguous in thenaturally occurring genome of the organism in which it originated. Forexample, an “isolated nucleic acid” may comprise a DNA molecule insertedinto a vector, such as a plasmid or virus vector, or integrated into thegenomic DNA of a prokaryotic or eukaryotic cell or host organism.Whenever applicable, the term “isolated nucleic acid” may also refer toa RNA molecule encoded by an isolated DNA molecule as defined above.Alternatively, the term may refer to an RNA molecule that has beensufficiently separated from other nucleic acids with which it would beassociated in its natural state (i.e. in cells or tissues). An “isolatednucleic acid” (either DNA or RNA) may further represent a moleculeproduced directly by biological or synthetic means and separated fromother components present during its production.

As used herein, the term “vector” is intended to mean a replicon, suchas a plasmid, cosmid, bacmid, phage or virus, to which another geneticsequence or element (either DNA or RNA) may be attached so as to bringabout the replication of the attached sequence or element.

As used herein, the terms “percent similarity”, “percent identity” and“percent homology” when referring to a particular sequence are used asset forth in the University of Wisconsin GCG software program.

As used herein, the term “substantially pure” is intended to refer to apreparation comprising at least 50-60% by weight of a given material(e.g., nucleic acid, oligonucleotide, protein, etc.). More preferably,the preparation comprises at least 75% by weight, and most preferably90-95% by weight of the given compound. Purity is measured by methodsappropriate for the given compound (e.g. chromatographic methods,agarose or polyacrylamide gel electrophoresis, HPLC analysis, and thelike). Described herein are substantially pure mutated RAF or KSRisoforms (e.g., nucleic acids, oligonucleotides, proteins, fragments,mutants, etc.).

As used herein, the term “oligonucleotide” is intended to sequences,primers and probes as described herein, and is defined as a nucleic acidmolecule comprised of two or more ribo- or deoxyribonucleotides,preferably more than three. The exact size of the oligonucleotide willdepend on various factors and on the particular application and use ofthe oligonucleotide.

As used herein, the term “primer” is intended to refer to anoligonucleotide, either RNA or DNA, either single-stranded ordouble-stranded, either derived from a biological system, generated byrestriction enzyme digestion, or produced synthetically which, whenplaced in the proper environment, is able to functionally act as aninitiator of template-dependent nucleic acid synthesis. When presentedwith an appropriate nucleic acid template, suitable nucleosidetriphosphate precursors of nucleic acids, a polymerase enzyme, suitablecofactors and conditions such as appropriate temperature and pH, theprimer may be extended at its 3′ terminus by the addition of nucleotidesby the action of a polymerase or similar activity to yield a primerextension product. The primer may vary in length depending on theparticular conditions and requirement of the application. For example,in diagnostic applications, the oligonucleotide primer is typicallyabout 20-40, or more nucleotides in length. The primer must be ofsufficient complementarity to the desired template to prime thesynthesis of the desired extension product, that is, to be able toanneal with the desired template strand in a manner sufficient toprovide the 3′ hydroxyl moiety of the primer in appropriatejuxtaposition for use in the initiation of synthesis by a polymerase orsimilar enzyme. It is not required that the primer sequence represent anexact complement of the desired template. For example, anon-complementary nucleotide sequence may be attached to the 5′ end ofan otherwise complementary primer. Alternatively, non-complementarybases may be interspersed within the oligonucleotide primer sequence,provided that the primer sequence has sufficient complementarity withthe sequence of the desired template strand to functionally provide atemplate-primer complex for the synthesis of the extension product.

As used herein, the term “probe” is intended to refer to anoligonucleotide, polynucleotide or nucleic acid, either RNA or DNA,whether occurring naturally as in a purified restriction enzyme digestor produced synthetically, which is capable of annealing with orspecifically hybridizing to a nucleic acid with sequences complementaryto the probe. A probe may be either single-stranded or double-stranded.The exact length of the probe will depend upon many factors, includingtemperature, source of probe and use of the method. For example, fordiagnostic applications, depending on the complexity of the targetsequence, the oligonucleotide probe typically contains about 20-40 ormore nucleotides in length, although it may contain fewer nucleotides.The probes herein are selected to be complementary to different strandsof a particular target nucleic acid sequence. This means that the probesmust be sufficiently complementary so as to be able to “specificallyhybridize” or anneal with their respective target strands under a set ofpre-determined conditions. Therefore, the probe sequence need notreflect the exact complementary sequence of the target. For example, anon-complementary nucleotide fragment may be attached to the 5′ or 3′end of the probe, with the remainder of the probe sequence beingcomplementary to the target strand. Alternatively, non-complementarybases or longer sequences can be interspersed into the probe, providedthat the probe sequence has sufficient complementarity with the sequenceof the target nucleic acid to anneal therewith specifically.

With respect to single-stranded nucleic acids, particularlyoligonucleotides, the term “specifically hybridizing” refers to theassociation between two single-stranded nucleotide molecules ofsufficiently complementary sequence to permit such hybridization underpre-determined conditions generally used in the art (sometimes termed“substantially complementary”). In particular, the term refers tohybridization of an oligonucleotide with a substantially complementarysequence contained within a single-stranded DNA molecule as describedherein, to the substantial exclusion of hybridization of theoligonucleotide with single-stranded nucleic acids of non-complementarysequence. Appropriate conditions enabling specific hybridization ofsingle-stranded nucleic acid molecules of varying complementarity arewell known in the art. For instance, one common formula for calculatingthe stringency conditions required to achieve hybridization betweennucleic acid molecules of a specified sequence homology is set forthbelow (Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratory Press):

T _(m)=81.5° C.+16.6 Log [Na+]+0.41 (% G+C)−0.63 (% formamide)−600/#bpin duplex

As an illustration of the above formula, using [Na+]=[0.368] and 50%formamide, with GC content of 42% and an average probe size of 200bases, the T_(m) is 57° C. The T_(m) of a DNA duplex decreases by 1-1.5with every 1% decrease in homology. Thus, targets with greater thanabout 75% sequence identity would be observed using a hybridizationtemperature of 42° C.

The stringency of the hybridization and wash depends primarily on thesalt concentration and temperature of the solutions. In general, tomaximize the rate of annealing of the probe with its target, thehybridization is usually carried out at salt and temperature conditionsthat are 20-25° C. below the calculated T_(m) of the hybrid. Washconditions should be as stringent as possible for the degree of identityof the probe for the target. In general, wash conditions are selected tobe approximately 12-20° C. below the T_(m) of the hybrid. With regard tothe nucleic acids as described herein, a moderate stringencyhybridization is defined as hybridization in 6×SSC, 5× Denhardt'ssolution, 0.5% SDS and 100 μg/ml denatured salmon sperm DNA at 42° C.and washed in 2×SSC and 0.5% SDS at 55° C. for 15 minutes. A highstringency hybridization is defined as hybridization in 6×SSC, 5×Denhardt's solution, 0.5% SDS and 100 μg/ml denatured salmon sperm DNAat 42° C., and washed in 1×SSC and 0.5% SDS at 65° C. for 15 minutes. Avery high stringency hybridization is defined as hybridization in 6×SSC,5× Denhardt's solution, 0.5% SDS and 100 μg/ml denatured salmon spermDNA at 42° C., and washed in 0.1×SSC and 0.5% SDS at 65° C. for 15minutes.

Alternatively, as used herein, the term “probe” is intended to mean acompound which is labeled with either a detectable label or an affinitytag, and which is capable of binding, either covalently ornon-covalently, to a RAF or KSR kinase domain. When, for example, theprobe is non-covalently bound, it may be displaced by a test compound.When, for example, the probe is bound covalently, it may be used to formcross-linked adducts, which may be quantified and inhibited by a testcompound.

As used herein, the term “isolated protein” or “isolated and purifiedprotein” is intended to refer to a protein produced by expression of anisolated nucleic acid molecule as described herein. Alternatively, thisterm may refer to a protein that has been sufficiently separated fromother proteins with which it would naturally be associated, so as toexist in “substantially pure” form. “Isolated” is not meant to excludeartificial or synthetic mixtures with other compounds or materials, orthe presence of impurities that do not interfere with the fundamentalactivity, and that may be present, for example, due to incompletepurification, or the addition of stabilizers.

As used herein, the term “amino acid” is intended to mean a radicalderived from the corresponding α-amino acid by eliminating the hydroxylof the carboxy group and one hydrogen of the .alpha.-amino group. Forexample, the terms Gln, Ala, Gly, Ile, Arg, Asp, Phe, Ser, Leu, Cys,Asn, and Tyr represent the residues of L-glutamine, L-alanine, glycine,L-isoleucine, L-arginine, L-aspartic acid, L-phenylalanine, L-serine,L-leucine, L-cysteine, L-asparagine, and L-tyrosine, respectively. AminoAcid residues are provided below:

Three and single letter abbreviations for α-amino acids used throughoutare as follows:

Amino acid. Abbreviation Abbreviation Alanine Ala A Arginine Arg RAspartic acid Asp D Asparagine Asn N Cysteine Cys C Glutamic acid Glu EGlutamine Gln Q Glycine Gly G Isoleucine Ile I Histidine His H LeucineLeu L Lysine Lys K Methionine Met M Phenylalanine Phe F Proline Pro PSerine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine ValV

As used herein, the term “subject” is intended to mean humans andnon-human mammals such as primates, cats, dogs, swine, cattle, sheep,goats, horses, rabbits, rats, mice and the like.

As used herein, the term “solid support” refers to any solid orstationary material to which reagents such as antibodies, antigens, andother test components can be attached. Examples of solid supportsinclude, without limitation, microtiter plates (or dish), microscope(e.g. glass) slides, coverslips, beads, cell culture flasks, chips (forexample, silica-based, glass, or gold chip), membranes, particles(typically solid; for example, agarose, sepharose, polystyrene ormagnetic beads), columns (or column materials), and test tubes.Typically, the solid supports are water insoluble.

As used herein, the term “instructional material” or a “user manual”includes a publication, a recording, a diagram, or any other medium ofexpression which can be used to communicate the usefulness of reagentsfor performing a method as described herein.

As used herein, the term “biological sample” is intended to refer to asubset of the tissues of a biological organism, its cells or componentparts (e.g. body fluids, including but not limited to, blood, mucus,lymphatic fluid, synovial fluid, cerebrospinal fluid, saliva, amnioticfluid, amniotic cord blood, urine, vaginal fluid and semen).

We have discovered, using a combination of structural analysis,site-directed mutagenesis and functional studies in vivo, a dimerizationinterface in RAF and KSR. We have identified a number of residues withinthe RAF and KSR kinase domains which, when mutated, prevent theformation of oncogenic dimers. Through this, we have discovered that RAFcatalytic function is regulated in response to a specific mode ofdimerization of its kinase domain (which we term the side-to-sidedimer). Furthermore, we have discovered that the RAF-relatedpseudo-kinase KSR also participates in forming side-to-side heterodimerswith RAF and thereby can trigger RAF activation. This mechanism providesan elegant explanation for the longstanding conundrum regarding RAFcatalytic activation and provides an explanation for the capacity ofKSR, despite lacking catalytic function, to directly mediate RAFactivation. We have also demonstrated that RAF side-to-side dimerformation is essential for aberrant signaling by oncogenic B-RAF mutantsand we have identified an oncogenic mutation that acts specifically bypromoting side-to-side dimer formation. These discoveries allow us toidentify the side-to-side dimer interface of RAF as a potentialtherapeutic target for intervention in B-RAF-dependent tumourigenesis.

I: Nucleic Acid Molecules, Vectors, Cells, Transgenes and TransgenicNon-Human Mammals

Described herein are mutated isoforms of RAF and KSR proteins.Furthermore, we have discovered that single point mutations in thedimerization interface of RAF or KSR kinase domains prevents theformation of side-to-side dimers, when compared to wild type RAF or KSR.The single point mutations in the RAF kinase domain are at residuesH449, G450, R481H, L487, F488, M489, Y538, A541 and K542 with themutations being H449E, G450W, R481H, L487R, F488A, F488L, M489W, Y538F,A541E and K542E. The single point mutations in the KSR kinase domain areat residues H699, G700, R732, L738, F739, M740, Y790, A793 and R794 withthe mutations being H699E, G700W, R732H, L738R, F739A, F739L, M740W,Y790F, A793E and R794E. We have also discovered that the isolated kinasedomain of RAF forms homodimers in aqueous solution. Similar behavior isexpected for the isolated KSR kinase domain as well as heterodimersshould form in aqueous solution upon mixing equimolar amounts of RAF andKSR kinase domains.

Thus, a substantially pure DNA molecule, such as genomic, cDNA, or asynthetic DNA molecule, encodes one of the mammalian or non-mammalianRAF or KSR isoforms in which one or more nucleotide substitutionshas/have been incorporated into the dimerization interface.

In certain embodiments, DNA sequences are substantially identical to theDNA sequences, or a fragment thereof, as illustrated in FIGS. 14Athrough 14F (SEQ ID NO's: 8, 10, 12, 14, 16, and 18). Another aspectfeatures RNA, which is encoded by the DNA described herein. In oneexample, the RNA is mRNA. In another example, the RNA is antisense RNA.

Also contemplated are oligonucleotide probes, which specificallyhybridize with the nucleic acid molecules as described herein. Incertain examples, the probe specifically hybridizes with mutated RAF orKSR nucleic acid molecules (e.g. a nucleic acid having a sequenceencoding a mutated RAF or KSR protein) while not hybridizing with thewild type or “normal” sequence under high or very high stringencyconditions. Primers capable of specifically amplifying mutated RAF orKSR encoding nucleic acids described herein are also contemplatedherein. As mentioned previously, such oligonucleotides are useful asprobes and primers for detecting, isolating or amplifying mutated RAF orKSR genes.

Nucleic acid molecules encoding the mutated RAF or KSR proteins, asdescribed herein, can be prepared by known general methods or isolatedfrom appropriate biological sources using methods known in the art.Additionally, cDNA or genomic clones having homology with human andother known mammalian RAF or KSR, for example, mouse, rat, and the like,or non-mammalian RAF or KSR, such as Drosophila, may be isolated fromother species using oligonucleotide probes corresponding topredetermined sequences within the human RAF or KSR encoding nucleicacids.

Nucleic acids described herein may be maintained as DNA in anyconvenient vector. Accordingly, vectors comprising a nucleic acidmolecule as described herein and more particularly a plasmid expressionvector are encompassed. Also encompassed are host cells transformed withsuch vectors and transgenic animals comprising such a nucleic acidmolecule as described herein. Those cells and animals could serve asmodels of disease in order to study the mechanism of the function of theRAF or KSR gene and also allow for the screening of therapeutics.

In some embodiments, the vector, host cell or transgenic animal comprisea nucleic acid molecule (a transgene) encoding a mutated RAF or KSRprotein that is expressed or delivered to tissues. The host cell is atransformed and stable cell line constitutively expressing the mutantRAF or KSR isoform.

Methods for producing host cells and transgenic animals are known in theart. Host cells include, but are not limited to mammalian, yeast orbacterial cells Transgenic animals can be selected from non-humanmammals such as farm animals (such as pigs, goats, sheep, cows, horses,rabbits, and the like), rodents (such as rats, guinea pigs, mice, andthe like), non-human primates (such as baboon, monkeys, chimpanzees, andthe like), and domestic animals (such as dogs, cats, and the like) andwild and domestic (such as swans, ducks, fowl and the like). Atransgenic animal is an animal having cells that contain a transgenewhich was introduced into the animal or an ancestor of the animal at aprenatal (embryonic) stage. The cells and transgenic animals can beuseful to identify mutated RAF or KSR proteins specific to each organ,and monitoring dimerization of the RAF and KSR in response totherapeutic treatment.

II: Mutated RAF or KSR Polypeptides

A mutated RAF or KSR polypeptide sequence may have 80% homology or morewith any of the amino acid sequences disclosed herein. A mutated RAF orKSR polypeptide sequence as described herein may also comprise at least50 or more contiguous amino acids of any of sequences disclosed herein.

Mutated dimer interface residues in Drosophila RAF or Drosophila KSR andtheir equivalent positions in mammalian B-RAF or KSR1 are provided inthe Tables below:

Hsap Mmus Dmel RAF BRAF BRAF (Acc. # (Acc. # (Acc. # P11346) P15056)P28028) H449E H477 H514 G450W G478 G515 R481H R509 R546 L487R L515 L552F488A F516 F553 F488L F516 F553 M489W M517 M554 Y538F Y566 Y603 A541EA569 A606 K542E K570 K607

Dmel KSR Hsap Mmus (Acc. # KSR1 (Acc. KSR1 (Acc. Q24171) # Q8IVT5) #Q61097) H699E H631 H583 G700W G632 G584 R732H R663 R615 L738R L669 L621F739A F670 F622 F739L F670 F622 M740W M671 M623 Y790F Y721 Y673 A793EA724 A676 R794E K725 K677

Other dimer interface residues in Drosophila RAF or Drosophila KSR andtheir equivalent positions in mammalian B-RAF or KSR1, and which aremutatable include those in the following Tables:

Hsap Mmus Dmel RAF BRAF BRAF (Acc. # (Acc. # (Acc. # P11346) P15056)P28028) E420 D448 D485 W422 W450 W487 W448 W476 W513 K478 R506 R543 K479K507 K544 T480 T508 T545 H482 H510 H547 C483 V511 V548 Q502 Q530 Q567D537 D565 D602 L560 L588 L625 S561 T589 T626 E687 E715 E752

Dmel KSR Hsap Mmus (Acc. # KSR1 (Acc. KSR1 (Acc. Q24171) # Q8IVT5) #Q61097) K670 Q602 Q554 W672 W604 W556 W698 W630 W582 K729 R660 R612 N730Q661 Q613 T731 T662 T614 H733 H664 H616 E734 E665 E617 S754 S685 S637G789 G720 G672 K812 K743 K695 V813 V744 V696 E941 E876 E828

SwissProt Accession Numbers are Provided for Reference

Referring to FIGS. 14A through 14F, specifically SEQ ID NO's: 7, 9, 11,13, 15 and 17, the amino acid positions for the experimentally verifieddimer interface residues are shaded in the protein sequences presented.In these Figures, predicted additional dimer interface residues areunderlined.

In some embodiments, the mutated RAF or KSR polypeptide is an isolatedmutated protein in which the mutations are located in the RAF or KSRkinase domain, specifically in the dimerization interface. In certainexamples, the mutated RAF polypeptide comprises one or more mutationsselected from H449E, G450W, R481H, L487R, F488A, F488L, M489W, Y538F,A541E and K542E. In certain examples, the mutated KSR polypeptidecomprises one or more mutations selected from H699E, G700W, R732H,L738R, F739A, F739L, M740W, Y790F, A793E and R794E.

Mutated RAF or KSR proteins or polypeptides as described herein may beprepared in a variety of ways, according to known methods. The proteinsmay be purified from appropriate sources, e.g., transformed bacterial oranimal cultured cells or tissues, by immunoaffinity purification. Theavailability of nucleic acid molecules encoding mutated RAF or KSRprotein enables production of the protein using in vitro expressionmethods and cell-free expression systems known in the art. In vitrotranscription and translation systems are commercially available, e.g.,from Promega or Invitrogen.

Alternatively, larger quantities of mutated RAF or KSR proteins orpolypeptides may be produced by expression in a suitable prokaryotic oreukaryotic system. For example, part or all of a DNA molecule encodingfor mutated RAF or KSR may be inserted into a plasmid vector adapted forexpression in a bacterial cell, such as E. coli. Such vectors comprisethe regulatory elements necessary for expression of the DNA in the hostcell positioned in such a manner as to permit expression of the DNA inthe host cell. Such regulatory elements required for expression includepromoter sequences, transcription initiation sequences and, optionally,enhancer sequences. Mutated RAF or KSR proteins or polypeptides producedby gene expression in a recombinant prokaryotic or eukaryotic system maybe purified according to methods known in the art.

Thus, another embodiment includes a method of producing a mammalianmutated RAF or KSR polypeptide includes providing a cell transformedwith a nucleic acid sequence encoding a mammalian mutated RAF or KSRpolypeptide positioned for expression in the cell. The mutated RAF orKSR polypeptide has an amino acid change at one of the positionsdepicted in FIGS. 14A through 14F (SEQ ID NO's: 7, 9, 11, 13, 15 and 17)that correspond to specific dimerization interface residues. Thetransformed cell is cultured under conditions for expressing the nucleicacid; which then produces the mammalian mutated RAF or KSR polypeptide.

A dominant-negative protein is a protein that antagonizes the action ofits normal counterpart. A dominant-negative RAF would be a mutant RAFthat prevents endogenous RAF from performing its natural (or oncogenic)function. Such a dominant-negative RAF (or KSR) protein could do so bysequestering away key proteins that normally act in concert withendogenous RAF (or KSR). For example, overexpression of akinase-defective RAF construct is known to act as a dominant-negative inpart by its ability to out-compete for endogenous RAS, which is normallycritical for RAF activation.

Thus a dominant negative mutant polypeptide of mammalian RAF or KSR,wherein the mutant polypeptide comprises a kinase domain having adisabled dimerization interface and therefore does not associate to a WTmammalian RAF or KSR dimerization interface.

The use of a dominant-negative polypeptide could be treating orpreventing a disease in a subject, in which the disease beingcharacterized by RAF/RAF homodimerization or RAF/KSR heterodimerization.This method comprises administering to the subject in need thereof, anexpression vector encoding mutated RAF or KSR polypeptide, the mutatedRAF or KSR polypeptide being positioned in the vector for expression ina cell of the subject in which RAF/RAF homodimerization or RAF/KSRheterodimerization is taking place, so as to treat or prevent thedisease.

III: Detection Methods

Recombinant WT and mutated RAF or KSR polypeptides can be used during invitro RAF or KSR dimerization experiments to follow the dimerization ofRAF or KSR protomers. RAF or KSR polypeptides mutants can also beco-transfected in mammalian cells with target protein substrates, suchas WT RAF or KSR.

Changes in WT and mutated RAF or KSR polypeptide dimerization inresponse to a potential therapeutic agent, and across cell phenotypes,can be monitored by measuring the variation of the levels ofphosphorylated MEK in the presence or absence of rapamycin in animalcells such as S2 cells.

The RAF or KSR dimerization appears to be involved in many aspects ofcancer from initiation to metastasis. One additional aspect includes amethod of detecting in a subject susceptibility to express mutant RAF orKSR polypeptide. The method includes taking a biological sample from thesubject that contains a sufficient amount of a nucleic acid, forexample, DNA, and sequencing predetermined regions of the DNA, whichencodes a RAF or KSR mutated polypeptide. By comparing this sequencewith a corresponding sequence from a non-susceptible control subject, aRAF or KSR mutation known to be indicative of the susceptibility can beidentified.

Thus, a method of detecting the presence of a mutation in a RAF kinasedomain or a KSR kinase domain, comprises a) providing a WT RAF kinasedomain or a WT KSR kinase domain and a suspected mutant RAF kinasedomain or a mutant KSR kinase domain, each domain having a cysteineresidue located at its N-terminus; b) incubating the WT RAF kinasedomain or the WT KSR kinase domain and the suspected mutant RAF kinasedomain or the suspected mutant KSR kinase domain with differentcross-linking detectable labels; c) incubating together equimolaramounts of the labelled WT RAF kinase domain or the labeled WT KSRkinase domain and detecting a signal from the detectable label so as toprovide a dimerization reference signal; and d) incubating equimolaramounts of the labeled suspected mutant B-RAF kinase domain or thesuspected mutant KSR kinase domain and detecting a signal from thedetectable labels, an absent signal or a reduce signal compared to thatof the dimerization reference signal being an indication that a mutantB-RAF kinase domain or a mutatent KSR kinase domain is present.

Also included is a bioluminescence resonance energy transfer (BRET)fusion molecule, and method of use. The fusion molecule comprises threecomponents: a bioluminescent donor protein (donor) and a fluorescentacceptor molecule (acceptor), wherein the acceptor can accept energyfrom the donor-generated luminescence when these components are in anappropriate spatial relationship and in the presence of an appropriatesubstrate. A modulator (a drug-like compound for example) can eitherinfluence the proximity/orientation of the donor and the acceptor andthereby the energy transfer between these components, or it can play adifferent role in affecting the energy transfer between thedonor-generated activated product and the acceptor.

Thus, there is provided a method of monitoring the formation of RAF/RAFor RAF/KSR kinase domain dimers to detect mutations inhibitingdimerization or drug-like molecules interfering with dimerization. Thismethod comprises a) fusing either (i) a RAF kinase domain or (ii) a KSRkinase domain at either of their N- or C-termini to a BRET donor or aBRET acceptor to produce donor labeled and acceptor labeled fusionproteins; b) expressing the fusion proteins to identify combinationsthat provide specific BRET signals; c) introducing dimer interfacemutations into either of the labeled fusion proteins; d) expressing thelabeled mutated fusion proteins with WT RAF or KSR kinase domains; e)measuring the BRET signals, a loss or significant reduction of the BRETsignal using dimer interface mutations as opposed to mutations remotefrom the interface, being an indication that a specific BRET signalwhich depends on the RAF/RAF or RAF/KSR dimerization interface has beenobtained.

In one example, the BRET donor is renilla luciferase variant II orrlucII and the BRET acceptor is GFP10. The acceptor label is YellowFluorescent Protein (YFP). The donor labeled fusion protein is SEQ IDNO's: 24, 34, 42 and 48, whereas the acceptor labeled fusion protein isSEQ ID NO's: 22, 30, 40 and 54. The donor labeled mutated fusionproteins are SEQ ID NO's: 36, 50 and the acceptor labeled mutated fusionproteins are SEQ ID NO: 32.

IV: Antibodies and Kits

Also provided are antibodies capable of immunospecifically binding tomutated RAF or KSR proteins and polypeptides as described herein. Suchantibodies may include, but are not limited to, polyclonal antibodies,monoclonal antibodies (mAbs), humanized or chimeric antibodies, singlechain antibodies, Fab fragments, F(ab′)2 fragments, fragments producedby a Fab expression library, anti-idiotypic (anti-Id) antibodies, andepitope-binding fragments of any of the above. Such antibodies may bemay be used for immunoaffinity enrichment of the mutated RAF or KSR orthey may be used in a kit for detecting in a subject the susceptibilityto develop a condition or an increased likelihood of developing acondition characterized by dimerization of RAF and/or KSR.

Polyclonal antibodies directed toward mutated RAF or KSR protein,polypeptides or fragments thereof may be prepared according to standardmethods. In one example, monoclonal antibodies are prepared, such thatantibodies react immunospecifically with predetermined epitopes of themutated RAF or KSR protein. In one example, the antibodies areimmunogically specific to mutated RAF or KSR proteins and polypeptides.Monoclonal antibodies may be prepared according to general methods knownin the art. Polyclonal or monoclonal antibodies that immunospecificallyinteract with mutant RAF or KSR proteins can be utilized for identifyingand purifying such proteins. For example, antibodies may be utilized foraffinity separation of proteins with which they immunospecificallyinteract. Antibodies may also be used to immunoprecipitate proteins froma sample containing a mixture of proteins and other biologicalmolecules.

One advantageous use of antibodies as described herein is in the use ofa kit for monitoring the RAF or KSR dimerization activity of a cell orthe binding of RAF or KSR to specific protein substrates such as the14-3-3 proteins. This information may be used for purposes of diagnosis,prognosis or for predicting the response to treatment. Examples ofdiseases include cancer. The kit comprises a substantially pure antibodythat specifically binds to a mammalian mutated RAF or KSR polypeptideand a means for detecting the binding of the antibody to the mammalianRAF or KSR polypeptide.

V: Screening Methods

Because we have identified the amino acid residues involved in RAF/RAFhomodimerization and RAF/KSR heterodimerization, we can use thisknowledge to screen for potential therapeutic agents which interact,either covalently or non-covalently, with the WT amino residuecounterparts. Thus, one additional aspect includes methods ofidentifying biological agents or small molecules that modulate orprevent RAF or KSR dimerization activity in the cell or modification ofthe regulation of protein RAF or KSR dimerization. This could also beexploited for example to screen for inhibitors, activators or modulatorsof RAF or KSR dimerization. The identified agents or molecules could beexploited as research reagents or for therapeutic purposes. The methodcould be used for in vitro screening assays using purified RAF or KSR WTpolypeptides.

Generally speaking, there is provided a method of identifying inhibitorsof RAF/RAF or RAF/KSR dimerization that bind to a RAF or KSR kinasedomain, the RAF or KSR full protein or the kinase domain is bound to asupport, and a potential inhibitor is added to the assay. Alternatively,the potential inhibitor may be bound to the support and the RAF or KSRfull protein or the kinase domain is added.

Additionally, the above described BRET assay can be used as a method ofidentifying a potential inhibitor of RAF/RAF homodimerization. Thismethod comprises a) fusing a RAF kinase domain at either of its N- orC-termini to a BRET donor or a BRET acceptor to produce donor labeledand acceptor labeled fusion proteins; b) expressing the fusion proteinsto identify combinations that provide specific BRET signals; c)introducing dimer interface mutations into either of the labeled fusionproteins; d) expressing the labeled mutated fusion proteins with WT RAFkinase domains; e) contacting the interface with the potentialinhibitor; and f) measuring the BRET signals, a loss or significantreduction of the BRET signal for the wild-type RAF/RAF BRET pair beingan indication that the inhibitor is specifically bound to the interface.

There are a number of ways in which to determine the binding of apotential inhibitor to the RAF or KSR kinase domain. In one way, thepotential inhibitor, for example, may be fluorescently or radioactivelylabeled and binding determined directly. For example, this may be doneby attaching the RAF or KSR full protein or the kinase domain to a solidsupport, adding a detectably labeled potential inhibitor, washing offexcess reagent, and determining whether the amount of the detectablelabel is present on the solid support. Numerous blocking and washingsteps may be used, which are known to those skilled in the art.

In some cases, only one of the components is labeled. For example,specific residues, such as those identified as described herein, in theRAF or KSR kinase domain may be labeled. Alternatively, more than onecomponent may be labeled with different labels; for example, using I¹²⁵for the RAF or KSR domain, and a fluorescent label for the potentialinhibitor.

As used herein, the terms “drug candidate”, “test compounds” or“potential inhibitor” are used interchangeably and describe anymolecule, for example, protein, oligopeptide, small organic molecule,polysaccharide, polynucleotide, and the like, to be tested forbioactivity. The compounds may be capable of inhibiting the formation ofRAF/RAF homodimers or RAF/KSR heterodimers.

Drug candidates can include various chemical classes, although typicallythey are small organic molecules having a molecular weight of more than100 and less than about 2,500 Daltons. Candidate agents typicallyinclude functional groups necessary for structural interaction withproteins, for example, hydrogen bonding and lipophilic binding, andtypically include at least an amine, carbonyl, hydroxyl, ether, orcarboxyl group. The drug candidates often include cyclical carbon orheterocyclic structures and/or aromatic or polyaromatic structuressubstituted with one or more functional groups.

Drug candidates can be obtained from any number of sources includinglibraries of synthetic or natural compounds. For example, numerous meansare available for random and directed synthesis of a wide variety oforganic compounds and biomolecules, including expression of randomizedoligonucleotides. Alternatively, libraries of natural compounds in theform of bacterial, fungal, plant and animal extracts are available orreadily produced. Additionally, natural or synthetically producedlibraries and compounds are readily modified through conventionalchemical, physical and biochemical means.

Competitive screening assays may be done by combining a RAF or KSRkinase domain and a labeled probe to form a probe:RAF or KSR kinasedomain complex in a first sample followed by adding a potentialinhibitor from a second sample. The binding of the potential inhibitoris determined, and a change or difference in binding between the twosamples indicates the presence of a test compound capable of binding tothe RAF or KSR kinase domain and potentially modulating the RAF or KSR'sdimerizing ability.

In one case, the binding of the potential inhibitor is determinedthrough the use of competitive binding assays. In this embodiment, theprobe is labeled with a fluorescent label. Under certain circumstances,there may be competitive binding between the potential inhibitor and theprobe. Potential inhibitors which displace the probe, resulting in achange in fluorescence as compared to control, are considered to bind tothe RAF or KSR kinase domain.

In one case, the potential inhibitor may be labeled. The potentialinhibitor is added first to the RAF or KSR domain for a time sufficientto allow binding to form a complex.

Formation of the probe:RAF or KSR domain complex typically requireincubations of between 4° C. and 40° C., for between 10 minutes to about1 hour to allow for high-throughput screening. Any excess of reagentsare generally removed or washed away. The potential inhibitor is thenadded, and the presence or absence of the labeled component is followed,to indicate binding to the RAF or KSR kinase domain.

In one case, the probe is added first, followed by the potentialinhibitor. Displacement of the probe is an indication the potentialinhibitor is binding to the RAF or KSR domain and thus is capable ofbinding to, and potentially modulating or inhibiting the dimerization ofRAF and KSR. Either component can be labeled. For example, the presenceof probe in the wash solution indicates displacement by the potentialinhibitor. Alternatively, if the potential inhibitor is labeled, thepresence of the probe on the support indicates displacement.

In one case, the potential inhibitor may be added first, with incubationand washing, followed by the probe. The absence of binding by the probemay indicate the potential inhibitor is bound to the RAF or KSR domainwith a higher affinity. Thus, if the probe is detected on the support,coupled with a lack of potential inhibitor binding, may indicate thepotential inhibitor is capable of binding to the RAF or KSR kinasedomain.

Modulation is tested by screening for a potential inhibitor's ability tomodulate the activity of RAF or KSR and includes combining a potentialinhibitor with a RAF or KSR kinase domain, as described above, anddetermining an alteration in the biological activity of RAF or KSR.Therefore in this case, the potential inhibitor should both bind to theRAF or KSR kinase domain (although this may not be necessary), and alterits biological activity as defined herein.

Positive controls and negative controls may be used in the assays. Allcontrol and test samples are performed multiple times to obtainstatistically significant results. Following incubation, all samples arewashed free of non-specifically bound material and the amount of boundprobe determined. For example, where a radiolabel is employed, thesamples may be counted in a scintillation counter to determine theamount of bound potential inhibitor.

Typically, the signals that are detected in the assay may includefluorescence, resonance energy transfer, time resolved fluorescence,radioactivity, fluorescence polarization, plasma resonance, orchemiluminescence and the like, depending on the nature of the label.Detectable labels useful in performing screening assays as describedherein include a fluorescent label such as Fluorescein, Oregon green,dansyl, rhodamine, tetramethyl rhodamine, texas red, Eu³⁺; achemiluminescent label such as luciferase; calorimetric labels;enzymatic markers; or radioisotopes such as tritium, I¹²⁵ and the like

Affinity tags, which may be useful in performing the screening assays asdescribed herein include biotin, polyhistidine and the like.

EXAMPLES

1. S2 Expression Plasmids

Copper-inducible pMet vectors were used for functional assays conductedin S2 cells as previously described (Douziech, M., Sahmi, M., Laberge,G. & Therrien, M. A KSR/CNK complex mediated by HYP, a novel SAMdomain-containing protein, regulates RAS-dependent RAF activation inDrosophila. Genes Dev 20, 807-19 (2006)), Roy, F., Laberge, G.,Douziech, M., Ferland-McCollough, D. & Therrien, M. KSR is a scaffoldrequired for activation of the ERK/MAPK module. Genes Dev 16, 427-38(2002)). The FRB-RAF^(K455S) fusion construct was assembled by insertingan AseI/NotI PCR fragment encompassing residues 328-738 of RAF into theAseI/NotI site of FRB-KSR (Roy, F., Laberge, G., Douziech, M.,Ferland-McCollough, D. & Therrien, M. KSR is a scaffold required foractivation of the ERK/MAPK module. Genes Dev 16, 427-38 (2002)). TheKSR-RAF chimera-A corresponds to KSR¹⁻⁶⁶⁵ fused to RAF⁴¹⁷⁻⁷³⁹, whereaschimera-B replaced the N-lobe of KSR (a.a. positions 666-757) with theone of RAF (a.a. positions 417-505). In both cases, the RAF N-lobecontained a K455M change to catalytically impair its kinase activity andthereby mimicked kinase-inert KSR. Variant full length Drosophila KSR,RAF or FRB/FKBP fusion mutants were generated by QuickChange mutagenesis(Stratagene). Mutagenized cDNAs were fully sequenced to verify that onlythe desired mutations had been introduced.

2. S2 Cell Assays

S2 cells were maintained in serum-free insect cell medium (Sigma) at 27°C. Cells were seeded at a density of 1.75×10⁶ cells/ml 24 h prior totransfection. Between 10 to 300 ng (or up to 900 ng for KSR_R732H) ofDNA was transfected per construct using Effectene (Qiagen). dsRNAs wereproduced and used in RNAi experiments as described (Roy, F., Laberge,G., Douziech, M., Ferland-McCollough, D. & Therrien, M. KSR is ascaffold required for activation of the ERK/MAPK module. Genes Dev 16,427-38 (2002)). Protein expression was induced by adding CuSO₄ (0.7 mM)36 h before harvesting the cells. For FRB/FKBP-mediated dimer formation,rapamycin (Sigma) was added (1 M) to the medium 2 h prior to harvestingthe cells. Lysates, immunoprecipitations, western blot procedures andantibodies were essentially as previously described (Douziech, M.,Sahmi, M., Laberge, G. & Therrien, M. A, KSR/CNK complex mediated byHYP, a novel SAM domain-containing protein, regulates RAS-dependent RAFactivation in Drosophila, Genes Dev, 20, 807-19 (2006)).

3. Bacterial Protein Expression and Purification

B-RAF (residues 448-723) and mutant series (R481H, L487R andL487R/E558K) were recombinantly expressed from pProEx (Invitrogen)plasmid in E. coli BL21 cells as TEV protease-cleavable 6× His-taggedfusions. To increase the level of soluble protein expression in E. coli,16 specific mutations (remote from the side-to-side dimer interface)were introduced in B-RAF as described Tsai, J. et al., Discovery of aselective inhibitor of oncogenic B-Raf kinase with potent antimelanomaactivity, Proc Natl Acad Sci, USA, 105, 3041-6 (2008). Expressedproteins were bound to Ni—NTA and eluted with imidazole and subjected toTEV protease treatment. Further purification was performed bysubtractive Ni—NTA and size exclusion (Superdex 200) chromatography.

4. Homology Modeling

A multiple sequence alignment of KSR and RAF kinase domains was used tobuild a structural model of the kinase domain of Drosophila KSR(residues 670-945) in SWISS-MODEL (Schwede, T., Kopp, J., Guex, N. &Peitsch, M. C. SWISS-MODEL: An automated protein homology-modelingserver. Nucleic Acids Res 31, 3381-5 (2003)). An initial model with atotal energy of −7474.3 KJ/mol was generated using the structure of thekinase domain of B-RAF as a template (chain A of PDB entry 1UWH) (Wan,P. T. et al., Mechanism of activation of the RAF-ERK signaling pathwayby oncogenic mutations of B-RAF, Cell, 116, 855-67 (2004)). This modelwas manually edited in COOT (Emsley, P. & Cowtan, K., Coot:model-building tools for molecular graphics, Acta Crystallogr D BiolCrystallogr, 60, 2126-32 (2004)) and a poorly modelled loop spanningresidues 821-838 was removed. To generate the KSR/RAF side-to-sideheterodimer, the modelled structure of KSR was superimposed onto chain Aof PDB entry 1UWH.

5. Analytical Ultracentrifugation

Equilibrium sedimentation was performed with a Beckman Optima XL-Aultracentrifuge and An60Ti rotor. B-RAF samples were prepared in 20 mMTris (pH 7.5), 200 mM NaCl, 5% glycerol and 1.5 mM TCEP for analysis.Data was collected at 4° C. for three protein concentrations (25 μM,12.5 μM, and 6.25 μM) at three rotor speeds (13,000 rpm, 18,000 rpm and23,000 rpm for B-RAF_wt and B-RAF_R481H or 12,000 rpm, 17,000 rpm and25,000 rpm for B-RAF_L487R and B-RAF-L487R/E558K). Model analysis of thedata was performed simultaneously in a global curve-fitting procedure(Origin software, Beckman). For this, data collected at 13,000 rpm and18,000 rpm for B-RAF_wt was analyzed at all three proteinconcentrations; data collected at 18,000 rpm and 23,000 rpm forB-RAF_R481H was analyzed at all three protein concentrations; datacollected at 17,000 rpm and 25,000 rpm for B-RAF_L487R was analyzed atall three protein concentrations; data collected at 17,000 rpm and25,000 rpm for B-RAF_L487R/E558K at 25 μM and 12.5 μM was analyzed. Theterm “global” refers to fits across all rotor speeds for a givenconcentration.

The global self association fit yielded an average molecular weight (MW)of 57,978 Da for B-RAF_wt. The ratio of the observed average MW to thetheoretical MW of the monomer is 1.9:1 suggesting that the samplecontains mostly dimers. A single-species dimer model (shown by the redline; FIG. 3C) best fit the observed data (blue circles; FIG. 3C),indicated by the random distribution of the residuals—a measure ofgoodness of fit (the residual is the difference between the observedvalue and the predicted value). For B-RAF_R481H, the global selfassociation fit yielded an average MW of 34,544 Da. The ratio of theobserved average MW to the theoretical MW of the monomer is 1.1:1suggesting that the sample contains mostly monomers. A single-speciesmonomer model (red line; FIG. 3C) best fit the observed data (bluecircles; FIG. 3C). For B-RAF_L487R, the global self association fityielded an average MW of 48,636 Da. The ratio of the observed average MWto the theoretical MW of the monomer is 1.5:1 suggesting that the samplecontains a mixture of monomers and dimers. Consistent with this, amonomer-dimer model (red line; FIG. 12) resulted in the best fit to theobserved data (blue circles; FIG. 12) with a dissociation constant (Kd)of 2 M (Note: In order to reliably estimate Kd values from an AUCexperiment, both species in a monomer-dimer equilibrium need to besufficiently represented in solution; in our AUC analyses, we observedsuch a monomer-dimer equilibrium only for the B-RAF_L487R dimer mutant).For B-RAF_L487R/E558K, the global self association fit yielded anaverage MW of 55,472 Da. The ratio of the observed average MW to thetheoretical MW of the monomer is 1.8:1 suggesting that the samplecontains mostly dimers and the data (blue circles; FIG. 12) was best fitto a single-species dimer model (red line; FIG. 12).

6. RNA Preparation and Quantitative Real-Time PCR (qPCR)

S2 cells were treated with specific RNAi for four days and total RNA wasextracted using Trizol reagent (Invitrogen) according to themanufacturer's instructions. qPCR analyses were performed as follows. Atotal of 2 μg of RNA was reverse transcribed using the High CapacitycDNA Archive Kit with random primers (Applied Biosystems, Foster City,Calif.) as described by the manufacturer.

Primer and probe sets from Universal ProbeLibrary were used forquantitative real-time PCR(https://www.roche-applied-science.com/sis/rtper/upl/index.jsp). Primerswere chosen so that the amplified regions did not overlap with the areastargeted by dsRNAs used for RNAi. PCR reactions for 384-well plateformats were performed using 2 μl of cDNA, 5 μl of the TaqMan fastUniversal PCR Master Mix (Applied Biosystems, CA), 2 μM of each primerand 1 μM of the Universal TaqMan probe in a total volume of 10 μl. TheABI PRISM® 7900HT Sequence Detection System (Applied Biosystems) wasused to detect the amplification level. The relative quantification oftarget genes was determined by using the CT method. Briefly, the Ct(threshold cycle) values of target genes were normalized to anendogenous control gene (Rp149) (CT=Ct target−Ct Rp149) and comparedwith a calibrator (wild type): CT=Ct_(Sample)−Ct_(Calibrator). Relativeexpression (RQ) or fold change was calculated using the SequenceDetection System (SDS) 2.2.2 software (Applied Biosystems) and theformula RQ=2^(−CT).

dsRNA primers: GFP amplicon (SEQ ID NO: 71)top 5′- CGTAAACGGCCACAAGTTCAG (SEQ ID NO: 72)bottom 5′- ACGAACTCCAGCAGGACCATG RAS amplicon (SEQ ID NO: 73)top 5′- AATACAAACTGGTCGTCGTTG (SEQ ID NO: 74)bottom 5′- AATCTACGATTCGGCTTGTTC CNK amplicon (SEQ ID NO: 75)top 5′- TTTGGACAGATCTATATGCAG (SEQ ID NO: 76)bottom 5′- TCGGTTCAAAGGTCTCCAG HYP amplicon (SEQ ID NO: 77)top 5′- CCGATTGTGTCACCCCTAAT (SEQ ID NO: 78)bottom 5′- CCACTTGAGCACATCGCTAA CK2α amplicon (SEQ ID NO: 79)top 5′- GACACTTCCTAGTGCGGCTCGCGTG (SEQ ID NO: 80)bottom 5′- GTAATCATACATCTGGTAATCTACC 14-3-3ε amplicon (SEQ ID NO: 81)top 5′- TGACTGAGCGCGAGAACAATG (SEQ ID NO: 82)bottom 5′- TCTTCTGCCTGCATATCGGAC 14-3-3ζ amplicon (SEQ ID NO: 83)top 5′- GACAGTCGATAAGGAAGAGCTGG (SEQ ID NO: 84)bottom 5′- TCGTTCAGTGTGTCCAGCTC

7. Screening Assays

Two independent assays were developed to monitor RAF dimerization. Thefirst is a FRET (fluorescence resonance energy transfer) assay. It isbased on the observation that bacterially-expressed human B-RAF kinasedomain form dimers in solution.

The second assay exploits the BRET (bioluminescence resonance energytransfer) technology to assess for RAF homodimerization or RAF-KSRheterodimerization using a cell-based system.

A. FRET Assay

A single cysteine residue is engineered at the N-terminus of thewild-type or mutant B-RAF kinase domain for cross-linking fluorescentprobes. Following bacterial expression and purification, independentbatch of proteins are labeled either with Alexa 555 (donor) or Alexa 647(acceptor). Labeled proteins are re-purified and then combined inequimolar ratios. FRET detection is carried out using a LuminescenceSpectrometer. Various controls are conducted in parallel. For instance,no FRET signal is detected when labeled proteins are tested alone.Similarly, no FRET signal or a significantly reduced one is detectedwhen dimer interface mutants (e.g. R481H-like) are tested in combinationwith wild-type B-RAF.

B. BRET Assay

The BRET assay can use either BRET1 or BRET2 as a means of measuringBRET signals. We used BRET2 donor (renilla luciferase variant II orrlucII) and acceptor (GFP10) fusions rather than BRET1 fusions (rluc andYFP) as well as the addition of a CAAX-box to target RAF to the plasmamembrane, since the BRET2 system is more sensitive and has a highersignal to noise ratio (Kocan, See et al., 2008), to independently fuseRAF and KSR kinase domains at either their N- or C-terminus. Theaddition of the CAAX-box is frequently used to generate BRAF gain offunction alleles and is reported in the literature (Leevers, Paterson etal., 1994). We focused on the human BRAF kinase domain (BRAF-KD)expressed from the pCDNA3.1 plasmid backbone (Invitrogen) in a HEK293transfection setup.

The assay included co-transfection of rlucII fused to the N-terminus ofthe human BRAF-KD (referred to below as the donor) and of N-terminallyGFP10-tagged hBRAF-KD (referred to below as the acceptor) both targetedto the plasma membrane with a CAAX-box. Transfections were performed inHEK293T cells with PEI as a transfection reagent in a 6-well format withvarying molar ratios of pCDNA3.1 donor:acceptor constructs (0:1, 0.25:1,0.5:1, 1:1, 2:1, 5:1, 10:1 and 20:1). 48 hours post transfection, thecells were washed and resuspended in tyrode buffer and transferred toopaque microtiter plates. GFP10 raw signal was read on a FlexStation 3plate reader (Molecular Devices) and BRET signals were read using aMithras LB 940 plate reader following the addition of DeepBlue C (DBC)at a concentration of 10 μM. The data was then analysed using theGraphPad Prism software package.

The assay was highly reproducible and the BRET ratio obtained for therlucII-BRAF-KD-CAAX versus GFP10-BRAF-KD-CAAX pair at saturatingconcentration of the acceptor construct was consistently between 4.7 and4.9.

We also generated the dimer interface mutation R481H of the BRAF-KD andmeasured its impact on the affinity of the BRAF-BRAF interaction. TheBRAF wt-wt pair yielded a significantly higher BRET ratio than when theR481H mutant was introduced as the donor or acceptor construct (FIG.15). This reduction was reproducible and significant in terms of boththe BRET_(max) and BRET₅₀ ratios (FIG. 15). This was indicative of asignificant decrease in BRAF-BRAF affinity when the dimer interface isperturbed.

Altogether, the BRET assay is specific for the BRAF-KD vs BRAF-KDinteraction and is highly sensitive to the genetic alteration of the nowwell-characterized dimer interface (Hatzivassiliou, Song et al.;Poulikakos, Zhang et al.; Rajakulendran, Sahmi et al. 2009).

All BRET assays were developed with the human B-RAF (hBRAF), C-RAF(hCRAF), KSR1 (hKSR1) and MEK1 (hMEK1) isoforms (see FIGS. 16 through 27and SEQ ID NO's: 19 through 70). In FIGS. 16 through 27, CDS stands forcoding sequences. And KD stands for kinase domain.

The mutagenised residues are labeled according to their position in theDrosophila orthologous protein sequence. Thus, the R481H mutation ofDrosophila RAF corresponds to the R509H mutation of human B-RAF, and theC922Y mutation of Drosophila KSR corresponds to the C722Y mutation ofhuman KSR1.

Results and Discussion

In Drosophila, RAF activation is regulated by a core complex thatnotably includes the proteins RAS, CNK, HYP and KSR amongst others(Claperon, A. & Therrien, M. KSR and CNK: two scaffolds regulatingRAS-mediated RAF activation, Oncogene, 26, 3143-58 (2007)). Of theseproteins, the function of KSR (Kinase Suppressor of Ras) in RAFactivation remains controversial. KSR contains a kinase domain ofclosest sequence similarity to RAF (Manning, G., Whyte, D. B., Martinez,R., Hunter, T. & Sudarsanam, S., The protein kinase complement of thehuman genome, Science, 298, 1912-34 (2002)) and was initially thought todrive RAF activation by virtue of its kinase activity. However,subsequent studies have been inconclusive in demonstrating this pointand thus relegated KSR as a pseudo-kinase (Boudeau, J.,Miranda-Saavedra, D., Barton, G. J. & Alessi, D. R., Emerging roles ofpseudokinases, Trends Cell Biol, 16, 443-52 (2006)). Because of itscapacity to bring MEK to RAF, the function of KSR is currentlyconsidered to be that of an organizing centre (or scaffold) in the ERKpathway (Kolch, W., Coordinating ERK/MAPK signalling through scaffoldsand inhibitors, Nat Rev Mol Cell Biol, 6, 827-37 (2005)).

We previously showed in Drosophila S2 cells that co-overexpression ofKSR with RAF and MEK stimulated RAF-dependent MEK phosphorylation(Douziech, M., Sahmi, M., Laberge, G. & Therrien, M., A KSR/CNK complexmediated by HYP, a novel SAM domain-containing protein, regulatesRAS-dependent RAF activation in Drosophila,. Genes Dev, 20, 807-19(2006)). If KSR was solely acting as a scaffold, we reasoned thatoverexpression of KSR without co-overexpression of its scaffold partnerswould perturb the optimal stoichiometry of KSR containing complexes withthe net effect of decreasing RAF activation. Since we observed increasedRAF activation, this suggested that KSR might possess an inherent RAFactivating capacity that becomes apparent upon overexpression. Toinvestigate whether KSR can stimulate increasing RAF activation in aconcentration dependent manner (as would be the case if KSR possessed anintrinsic RAF activating capacity), we titrated in increasing amounts ofKSR in S2 cells and monitored the effect by assessing RAF-dependent MEKphosphorylation (Roy, F., Laberge, G., Douziech, M., Ferland-McCollough,D. & Therrien, M., KSR is a scaffold required for activation of theERK/MAPK module, Genes Dev, 16, 427-38 (2002)). As shown in FIG. 1A,increasing levels of KSR correspondingly increased MEK phosphorylation.Surprisingly, this KSR-dependent RAF activation was unperturbed byRNAi-mediated knockdown of RAS (FIG. 1A; see FIG. 13A for ademonstration of the activity and specificity of the RAS dsRNA).Moreover, co-overexpression of a constitutively active RAS (RAS^(V12))under these conditions did not considerably augment MEK phosphorylation,suggesting that KSR can drive RAF activation independently of RASactivity when overexpressed in S2 cells (FIG. 1A). These results suggesta role for KSR in RAF activation beyond a scaffold-only function.

To more rigorously rule out a scaffolding function as the origin of theobserved stimulatory effect of overexpressed KSR on RAF, we used RNAi toknockdown a subset of known scaffold partners of KSR. Interestingly, inthis context RAF activation was also unperturbed by RNAi-mediatedknockdown of CNK or HYP (also known as AVE), which are normally requiredunder physiological conditions (Douziech, M., Sahmi, M., Laberge, G. &Therrien, M., A KSR/CNK complex mediated by HYP, a novel SAMdomain-containing protein, regulates RAS-dependent RAF activation inDrosophila, Genes Dev, 20, 807-19 (2006)), Douziech, M. et al., Bimodalregulation of RAF by CNK in Drosophila, Embo J, 22, 5068-78 (2003)) andRoignant, J. Y., Hamel, S., Janody, F. & Treisman, J. E., The novel SAMdomain protein Aveugle is required for Raf activation in the DrosophilaEGF receptor signaling pathway, Genes Dev, 20, 795-806 (2006) (FIG. 1B).Studies with mammalian cells recently suggested that CK2 (bound to KSR1)phosphorylates and activates B-RAF/C-RAF (Ritt, D. A. et al., CK2 is acomponent of the KSR1 scaffold complex that contributes to Raf kinaseactivation, Curr Biol, 17, 179-84 (2007)). We found that KSR not onlypotently stimulated RAF activation in the presence of RNAi-mediatedknockdown of CK2 (FIG. 1C), but that mutating the proposed CK2 bindingsite on KSR or the sites of CK2-mediated phosphorylation on RAF (Ritt,D. A. et al., CK2 Is a component of the KSR1 scaffold complex thatcontributes to Raf kinase activation, Curr Biol, 17, 179-84 (2007)) hadno impact on the ability of KSR to drive RAF activation under ouroverexpression conditions (FIG. 1C). Taken together, these resultssuggest that the overexpression of KSR in S2 cells unmasks an inherentactivation potential on RAF beyond its well established role as ascaffold.

We previously showed that the capacity of KSR to bind MEK was requiredfor the ability of RAF to phosphorylate MEK (Roy, F., Laberge, G.,Douziech, M., Ferland-McCollough, D. & Therrien, M., KSR is a scaffoldrequired for activation of the ERK/MAPK module, Genes Dev, 16, 427-38(2002)). This result supported a scaffolding role for KSR in RAFactivation. More recently, we identified a mutation in KSR (R732H)within its kinase domain that completely abolished its RAF activatingcapacity yet fully retained its ability to bind MEK and RAF Douziech,M., Sahmi, M., Laberge, G. & Therrien, M, A KSR/CNK complex mediated byHYP, a novel SAM domain-containing protein, regulates RAS-dependent RAFactivation in Drosophila, Genes Dev, 20, 807-19 (2006) (FIG. 2). Thismutant provided a starting point for unraveling the mechanism by whichKSR directly activates the catalytic function of RAF.

Since the kinase domain of KSR is most similar to that of RAF (Manning,G., Whyte, D. B., Martinez, R., Hunter, T. & Sudarsanam, S., The proteinkinase complement of the human genome, Science, 298, 1912-34 (2002), wehypothesized that the previously determined crystal structure of thekinase domain of human B-RAF (the human orthologue of Drosophila RAF)might provide a good model to discern the mechanism of action of the KSRR732H mutation. Indeed, Arg732 is not only invariant across all KSRproteins, it is invariant across the larger RAF/KSR family (but not inother closely related kinases; FIG. 4). Intriguingly, while thestructure of the kinase domain of B-RAF was reported as a monomer (Wan,P. T. et al., Mechanism of activation of the RAF-ERK signaling pathwayby oncogenic mutations of B-RAF, Cell, 116, 855-67 (2004)), theasymmetric unit of the crystal in fact contains two RAF kinase domainsthat interact in a unique side-to-side fashion involving the N-lobe oftheir kinase domains (FIG. 6A). This mode of dimerization, which was notappreciated to date, was observed in a total of five subsequent RAFstructure analyses (Wan, P. T. et al., Mechanism of activation of theRAF-ERK signaling pathway by oncogenic mutations of B-RAF, Cell, 116,855-67 (2004)), (Hansen, J. D. et al., Potent and selectivepyrazole-based inhibitors of B-Raf kinase, Bioorg Med Chem Lett, 18,4692-5 (2008)) (in distinct crystal lattices), suggesting that the modeof dimerization/oligomerization is functionally relevant rather than anartifact of crystal packing (FIG. 6B). Side-to-side dimerization of theRAF kinase domain buries a large surface area (˜1280 Å²) andprovocatively involves helix αC, a key structural element whoseconformation serves a regulatory function in numerous protein kinases(Huse, M. & Kuriyan, J. The conformational plasticity of proteinkinases. Cell 109, 275-82 (2002)) (FIG. 6A). Most notably, a specificmode of dimerization involving helix αC underlies an allostericmechanism for kinase activation for both PKR (Dar, A. C., Dever, T. E. &Sicheri, F. Higher-order substrate recognition of eIF2alpha by theRNA-dependent protein kinase PKR. Cell 122, 887-900 (2005)) and EGFR(Zhang, X., Gureasko, J., Shen, K., Cole, P. A. & Kuriyan, J. Anallosteric mechanism for activation of the kinase domain of epidermalgrowth factor receptor. Cell 125, 1137-49 (2006)) kinase domains (FIG.6C). As the structure of the RAF kinase domain adopts a productiveconformation in the dimeric crystal configuration, we reasoned thatside-to-side dimerization itself might directly modulate the attainmentof an active kinase conformation of RAF.

Projection of KSR/RAF conserved residues onto the RAF crystal structurerevealed that nearly the entire side-to-side dimer contact surface ofRAF, but no other surfaces, are conserved across the larger KSR/RAFfamily (FIG. 3A; FIG. 4). This suggested that KSR might form ananalogous dimer structure. Moreover, the position of Arg481 (theequivalent of Arg732 in KSR; FIG. 4) at the center of the side-to-sidedimer interface of the B-RAF crystal structure (FIG. 3B) hinted at thebasis by which the mutation of Arg732 in KSR might exert a functionaleffect by perturbing dimerization (for simplicity, we used theDrosophila RAF numbering scheme for discussion of human B-RAF positions;see the Table below for list of residue equivalence between B-RAF andDrosophila RAF).

Drosophila RAF Human B-RAF Trp422 Trp450 Trp448 Trp476 His449 His477Gly450 Gly478 Lys478 Arg506 Lys479 Lys507 Thr480 Thr508 Arg481 Arg509His482 His510 Cys483 Val511 Leu487 Leu515 Phe488 Phe516 Met489 Met517Gln502 Gln530 Asp537 Asp565 Tyr538 Tyr566 Ala541 Ala569 Lys542 Lys570Glu558 Glu586 Leu560 Leu588 Ser561 Thr589 Glu687 Glu715

In order to investigate the potential of the RAF kinase domain to formdimers in solution, we performed analytical ultracentrifugationexperiments (FIG. 3C). Equilibrium sedimentation analysis confirmed thatRAF can form dimers under the conditions tested (i.e., micromolarconcentrations). Consistent with the mode of dimerization seen in thecrystal structure, mutation of Arg481 in B-RAF converted it to apredominant monomer in solution. This result shows that the side-to-sidedimer configuration of RAF visualized in the crystal environments isalso sampled in solution. Based on these findings, we reasoned that theR732H mutation in KSR most likely perturbs KSR's ability to form ananalogous side-to-side homodimer or to form a side-to-side heterodimerwith RAF. This in turn could explain the mechanism by which theKSR_R732H mutation abolishes RAF activation.

If KSR mediates RAF activation by a mechanism involving the formation ofa specific side-to-side homodimer with itself (i.e. KSR/KSR side-to-sidehomodimer) or a heterodimer with the kinase domain of RAF (i.e. KSR/RAFside-to-side heterodimer), then mutation of other dimer interfaceresidues on KSR in close vicinity to Arg732 might also impair RAFactivation. Using our minimal KSR/RAF/MEK co-overexpression activationassay, we found this to be the case. Specifically, individual mutationof four additional residues (G700W, F739A, M740W and Y790F) on KSRseverely impeded its ability to induce RAF activation (FIG. 5A). If KSRmediates RAF activation by forming a specific side-to-side heterodimerwith the kinase domain of RAF, then mutations of the correspondingpositions (residues) on RAF should also impair RAF activation. As shownin FIG. 5A, we also found this to be the case. In contrast, controlmutations remote from the side-to-side dimer interface on the kinasedomains of both KSR and RAF showed no significant effect on RAFactivation (FIG. 5A). We note that none of the dimer interface mutationsin KSR detectably affected the KSR/MEK interaction, indicating that themutations did not simply destroy protein fold (data not shown). Theseresults confirm that the integrity of the side-to-side dimer interfaceon KSR and on RAF is essential for RAF activation.

While our results above are consistent with the possibility that KSR andRAF heterodimerize through their kinase domains, it is equally possiblethat KSR/KSR side-to-side homodimers might instead contribute to RAFactivation. To demonstrate that the formation of side-to-side kinasedomain heterodimers by KSR and RAF per se leads to RAF activation, weemployed the FRB/FKBP fusion protein system to inducibly promote KSR/RAFside-to-side heterodimer formation by the addition of rapamycin in vivo(Muthuswamy, S. K., Gilman, M. & Brugge, J. S. Controlled dimerizationof ErbB receptors provides evidence for differential signaling by homo-and heterodimers, Mol Cell Biol, 19, 6845-57 (1999)).

Towards this end, we fused a region encompassing the minimal kinasedomains of KSR and RAF to the FRB and FKBP fragments, respectively (Roy,F., Laberge, G., Douziech, M., Ferland-McCollough, D. & Therrien, M.,KSR is a scaffold required for activation of the ERK/MAPK module, GenesDev, 16, 427-38 (2002)) (See FIG. 5B for schematic). The use of theFRB/FKBP fusion in conjunction with a myristoylation signal on the FKBPfusion construct (to localize it to the membrane) allowed us to tightlymodulate heterodimerization of the kinase domains in arapamycin-dependent manner (Roy, F., Laberge, G., Douziech, M.,Ferland-McCollough, D. & Therrien, M., KSR is a scaffold required foractivation of the ERK/MAPK module, Genes Dev, 16, 427-38 (2002)). Inthis setup, we observed that promoting the KSR/RAF heterodimer byaddition of rapamycin was indeed sufficient to potently activate RAF asevidenced by the elevated levels of phosphorylated MEK (FIG. 5B). RAFactivation was selectively perturbed by ten specific mutations at theside-to-side dimer interface on both KSR (H699E, G700W, R732H, L738R,F739A, F739L, M740W, Y790F, A793E and R794E) and on RAF (H449E, G450W,R481H, L487R, F488A, F488L, M489W, Y538F, A541E and K542E), but not bycontrol mutations outside the side-to-side dimer interface of KSR or RAF(FIG. 5B; FIG. 8). Taken together, these results indicate that formationof the side-to-side heterodimer between KSR and RAF kinase domains isboth sufficient and necessary for RAF activation under the conditionstested.

As both RAF and KSR likely form identical side-to-side dimers, by virtueof having near identical dimerization surfaces (FIG. 3A), it isconceivable that both KSR/RAF heterodimers and RAF/RAF homodimers mightequally promote RAF activation, assuming RAF activation and downstreamsignaling is solely dependent on forming the kinase domain side-to-sidedimer. To investigate whether the RAF/RAF homodimers can also lead toRAF activation, we used the FRB/FKBP/rapamycin system to driveside-to-side homodimer formation of RAF kinase domains in vivo (see FIG.5C for schematic). To ensure our interpretation of side-to-side dimerformation induced activation is not confounded by transautophosphorylation activity within the RAF/RAF homodimer, we introduceda mutation (K455S) in the FRB-RAF fusion to catalytically impair itskinase activity (i.e. to effectively mimic the kinase dead state ofKSR). As shown in FIG. 5C, rapamycin induced formation of RAF/RAFhomodimers can indeed drive RAF activation in a manner dependent on theability to form the side-to-side dimers (FIG. 5C).

Although RAF/RAF homodimers are competent for activation, the level ofactivation is not as robust as that resulting from KSR/RAF heterodimers(based on quantification of induced MEK phosphorylation levels in thepresence and absence of rapamycin; not shown). If the side-to-side dimersurfaces are in fact functionally equivalent on both KSR and RAF, thisobservation suggests that the KSR kinase domain may have a secondfunction that is not shared with RAF. Based on the fact that KSR canstably bind MEK while RAF cannot (Roy, F., Laberge, G., Douziech, M.,Ferland-McCollough, D. & Therrien, M. KSR is a scaffold required foractivation of the ERK/MAPK module. Genes Dev 16, 427-38 (2002)), wereasoned that this may be the root of the difference. Since theside-to-side dimerization surface is comprised mainly by the N-lobe ofKSR and RAF kinase domains, and MEK binding function is criticallydependent on the C-lobe of KSR (Roy, F., Laberge, G., Douziech, M.,Ferland-McCollough, D. & Therrien, M., KSR is a scaffold required foractivation of the ERK/MAPK module, Genes Dev, 16, 427-38 (2002)), then aRAF N-lobe—KSR C-lobe chimera might possess both essential functions ofthe KSR kinase domain. If true, one would predict that substitution ofthe N-lobe of RAF into KSR, but not the whole kinase domain of RAF intoKSR, would lead to the maintenance of KSR's ability to promote RAFmediated phosphorylation of MEK. This indeed proved to be the case. Asshown in FIG. 7, overexpression of a form of KSR with a full kinasedomain swap with RAF (FIG. 7A, Chimera-A) poorly activated RAF, whileoverexpression of a form with just an N-lobe swap (FIG. 7A, Chimera-B)was as potent as wild type KSR in promoting MEK phosphorylation by RAF(FIG. 7B). Confirming that MEK binding is indeed constrained to theC-lobe of KSR, Chimera-B but not Chimera-A bound to MEK as assessed byco-immunoprecipitation (FIG. 7B). Taken together, these resultshighlight two distinct functions for the kinase domain of KSR in RAFsignaling. Firstly, the kinase domain of KSR functions as a scaffoldwhereby it binds to MEK and recruits it to RAF (i.e. KSR mediates RAFsubstrate targeting). Secondly, the kinase domain of KSR forms aside-to-side heterodimer with the kinase domain of RAF that underlies anallosteric mechanism for RAF catalytic activation.

Recent studies with mammalian cells, where multiple RAF isoforms exist,have found that RAF activation can also occur upon the physicaljuxtaposition of two isoforms of RAF mediated by 14-3-3 proteins (Weber,C. K., Slupsky, J. R., Kalmes, H. A. & Rapp, U. R., Active Ras inducesheterodimerization of cRaf and Braf., Cancer Res, 61, 3595-8 (2001)),(Rushworth, L. K., Hindley, A. D., O'Neill, E. & Kolch, W., Regulationand role of Raf-1/B-Raf heterodimerization, Mol Cell Biol, 26, 2262-72(2006)). Intriguingly, this activation route is independent of aphospho-transfer mechanism as reflected by the fact that in such RAF/RAFheterodimers, a kinase-dead isoform of RAF can activate a wild-typeisoform of RAF (Chen, C., Lewis, R. E. & White, M. A., IMP modulatesKSR1-dependent multivalent complex formation to specify ERK1/2 pathwayactivation and response thresholds, J Biol Chem, 283, 12789-96 (2008)).This behaviour is highly reminiscent of how KSR activates RAF. Wereasoned that 14-3-3 proteins, which are intrinsically dimeric, act topromote the specific side-to-side dimer conformation we see in the RAFcrystal structure in a manner analogous to our forced FRB-RAF/FKBP-RAFsystem (FIG. 5C). Consistent with this possibility, our modeling studiesshowed that the binding of dimeric 14-3-3 proteins concurrently to theC-terminal extension of two RAF kinase domains is fully compatible withthe adoption of a side-to-side dimer configuration (FIG. 10).

Interestingly, the 14-3-3 consensus binding site in human RAF isconserved in both RAF and KSR molecules in fly and in other organisms(FIG. 9A), suggesting that 14-3-3 could also act to promote RAFhomodimers and more potent KSR/RAF heterodimers in flies. Demonstratingthat 14-3-3 is indeed relevant for RAF activation in flies, we foundthat depletion of endogenous 14-3-3 proteins perturbed KSR-dependent RAFactivation (FIG. 9B). Consistent with the notion that 14-3-3 mediatesdimerization of KSR with RAF, mutation of the consensus 14-3-3 site inboth KSR and RAF impaired RAF activation (FIG. 9B). These resultssuggest that 14-3-3 proteins might act to promote specific KSR/RAF andRAF/RAF side-to-side kinase domain dimers.

Together, our study indicates that dimerization of the RAF kinase domainwith KSR or with other RAF molecules is central to its activationmechanism. We posit that other regulatory events that impinge on RAFactivation may also act by modulating dimerization. In this regard, thelarge group of scaffolding proteins that act together with RAF and KSR,such as 14-3-3 proteins, may serve to spatially and temporally regulatethe formation of side-to-side dimers (Douziech, M., Sahmi, M., Laberge,G. & Therrien, M. A, KSR/CNK complex mediated by HYP, a novel SAMdomain-containing protein, regulates RAS-dependent RAF activation inDrosophila, Genes Dev, 20, 807-19 (2006)), Garnett, M. J., Rana, S.,Paterson, H., Barford, D. & Marais, R., Wild-type and mutant B-RAFactivate C-RAF through distinct mechanisms involving heterodimerization,Mol Cell, 20, 963-9 (2005)), Rushworth, L. K., Hindley, A. D., O'Neill,E. & Kolch, W., Regulation and role of Raf-1/B-Raf heterodimerization,Mol Cell Biol, 26, 2262-72 (2006)), Chen, C., Lewis, R. E. & White, M.A., IMP modulates KSR1-dependent multivalent complex formation tospecify ERK1/2 pathway activation and response thresholds, J Biol Chem,283, 12789-96 (2008)). Moreover, the fact that the formation of B-/C-RAFheterodimers appears to depend on RAS activity (Garnett, M. J., Rana,S., Paterson, H., Barford, D. & Marais, R., Wild-type and mutant B-RAFactivate C-RAF through distinct mechanisms involving heterodimerization,Mol Cell, 20, 963-9 (2005)), strongly suggests that RAS may also play arole in forming side-to-side kinase domain dimers. In the absence ofRTK/RAS activation, a regulatory element in the N-terminus of RAFengages the C-terminal kinase domain to inhibit catalytic activity by anunknown mechanism (Chong, H. & Guan, K. L., Regulation of Raf throughphosphorylation and N terminus-C terminus interaction, J Biol Chem, 278,36269-76 (2003)). We reason that this autoinhibitory interaction mayinterfere with the ability of the kinase domain to adopt a productivedimer configuration.

Although dependent on many more components, the activation mechanism ofRAF appears analogous in principle, if not execution, to those employedby the PKR and EGFR protein kinases. In the case of the eIF2 proteinkinase PKR, the attainment of a specific dimer configuration by thekinase domain is regulated by the binding of dsRNA viral by-products toregions N-terminal to the kinase domain (Dar, A. C., Dever, T. E. &Sicheri, F., Higher-order substrate recognition of eIF2alpha by theRNA-dependent protein kinase PKR, Cell, 122, 887-900 (2005)), Dey, M. etal., Mechanistic link between PKR dimerization, autophosphorylation, andeIF2alpha substrate recognition, Cel, 122, 901-13 (2005). In the case ofEGFR kinase, adoption of a unique dimer/oligomer configuration by itskinase domain is regulated by the binding of growth factors to theextracellular ligand binding domain of the receptors (Zhang, X.,Gureasko, J., Shen, K., Cole, P. A. & Kuriyan, J., An allostericmechanism for activation of the kinase domain of epidermal growth factorreceptor, Cell, 125, 1137-49 (2006)) (FIG. 6C). Reflecting theimportance of self interaction in the function of all three proteinkinase families, residues comprising the self interaction surfaces ofthe kinase domain in addition to the catalytic infrastructure areevolutionarily conserved within each kinase family. In this regard, KSRis essentially equivalent to a RAF molecule. In effect, we reason thatRAF and KSR evolved from a single ancestral progenitor, one thatpossessed both protein kinase catalytic activity and stable substrate(MEK) binding function. Following a gene duplication event (Claperon, A.& Therrien, M., KSR and CNK: two scaffolds regulating RAS-mediated RAFactivation, Oncogene, 26, 3143-58 (2007)), one gene dispensed withphospho-transfer function (i.e. KSR) and the other dispensed with theability to stably bind MEK substrate (i.e. RAF). However, bothmaintained the ability to form allosteric dimers and this selectivepressure maintained the side-to-side dimer interface and interdependencebetween KSR and RAF proteins in ERK signaling.

The mapping of human cancer causing mutations to the activation segmentof B-RAF proved unequivocally that the activation segment of RAF is alsoa key modulator of its catalytic function (Wan, P. T. et al., Mechanismof activation of the RAF-ERK signaling pathway by oncogenic mutations ofB-RAF, Cell, 116, 855-67 (2004)), (Davies, H. et al., Mutations of theBRAF gene in human cancer, Nature, 417, 949-54 (2002)). Consistent withthis, we previously found that a mutation in the activation segment ofDrosophila RAF (RAF-AL^(ED)) strongly hyperactivated its catalyticactivity (Douziech, M., Sahmi, M., Laberge, G. & Therrien, M., A KSR/CNKcomplex mediated by HYP, a novel SAM domain-containing protein,regulates RAS-dependent RAF activation in Drosophila, Genes Dev, 20,807-19 (2006)), suggesting that it likely acts via a similar mechanismas those identified in human cancers (Wan, P. T. et al., Mechanism ofactivation of the RAF-ERK signaling pathway by oncogenic mutations ofB-RAF, Cell, 116, 855-67 (2004)). This raises the question of how kinasedomain dimerization and the modulation of activation segmentconformation are coordinated. Both events may be essential for thetransmission of a downstream signal or each event may be sufficient onits own. If both are essential, then oncogenic activation segmentmutants of RAF should still be sensitive to dimer interface mutations.Suggesting that this in fact is the case, introduction of a mutation(R481H) within the side-to-side dimer interface in Drosophila RAFeffectively nullifies the aberrant signaling properties of RAF-AL^(ED)(FIG. 11A).

Intriguingly, while most oncogenic RAF mutations act through modulationof the activation segment, one particular mutation, RAF E558K (E586K inhuman B-RAF), is located on the opposite surface of the kinase domainfrom the activation segment (Wan, P. T. et al., Mechanism of activationof the RAF-ERK signaling pathway by oncogenic mutations of B-RAF, Cell,116, 855-67 (2004)) and its mechanism of kinase activation remainedenigmatic. Most conspicuously, Glu558 lies on the side-to-side dimerinterface (FIG. 11B). If dimerization is indeed critical for RAFactivation, we questioned whether RAF_E558K might promote kinaseactivity by promoting dimerization. We reasoned that mutation of Glu558to the longer Lys (E558K) could potentially introduce a hydrogen bondwith Ser561 (conservative Thr589 in B-RAF) on the second RAF protomerthereby promoting dimer formation (FIG. 11B). Indeed, as tested below wefound that the RAF_E558K mutation promoted kinase domain dimerization insolution. Wild type RAF kinase domain is predominantly a dimer insolution (at the micromolar concentrations tested), which prevented adirect test of the RAF_E558K mutant for enhanced dimerization potential(FIG. 3C). To circumvent this problem, we employed the RAF_L487R dimermutant which displayed a weak monomer-dimer binding equilibrium insolution (FIG. 12).

Thus, introduction of the E558K mutation (RAF_L487R/E558K double mutant)transitioned RAF_L487R back to a predominantly dimeric state (FIG. 6).To investigate how the RAF_E558K mutation functions to hyperactivate RAFin vivo, we used the FRB/FKBP/rapamycin system to assess RAF activationin S2 cells. When the E558K mutation was introduced in the kinase-dead(K455S) background (FRB-RAF_K455S/E558K double mutant), it displayed noactivity when tested alone (not shown), but strongly hyperactivated theFKBP-RAF counterpart in a rapamycin dependent manner (FIG. 11C). Takentogether, the ability of the E558K mutant to act in trans (i.e. in thecontext of a kinase dead mutant) in vivo, and the ability of the E558Kmutation to promote kinase domain dimerization in vitro stronglysuggests that the mechanism by which the oncogenic RAF_E558K mutationacts is by promoting side-to-side dimers. Givern these results, it isnow possible to develop small molecules strategies that are directed atpreventing the formation of side-to-side dimers by RAF and which canserve as a therapeutic for RAF-dependent human tumors, one that wouldcomplement conventional strategies currently directed at inhibiting RAFenzymatic activity by blocking the catalytic cleft (Wu, S., Guo, W. &Fang, B., Development of small-molecule inhibitors of raf, RecentPatents Anti-Infect Drug Disc, 1, 241-6 (2006)).

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the present discovery and scope of the appendedclaims.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

1. A composition comprising: an aqueous solution of RAF/RAF homodimer.2-4. (canceled)
 5. A composition comprising: an aqueous solution ofRAF/KSR heterodimer. 6-40. (canceled)
 41. A method of detecting thepresence of a mutation in a RAF kinase domain, the method comprising: a)providing a WT RAF kinase domain and a suspected mutant RAF kinasedomain, each domain having a cysteine residue located at its N-terminus;b) incubating the WT RAF kinase domain and the suspected mutant RAFkinase domain with different cross-linking detectable labels; c)incubating together equimolar amounts of the labeled WT RAF kinasedomain and detecting a signal from the detectable label so as to providea dimerization reference signal; and d) incubating equimolar amounts ofthe labeled suspected mutant B-RAF kinase domain and detecting a signalfrom the detectable labels, an absent signal or a reduce signal comparedto that of the dimerization reference signal being an indication that amutant B-RAF kinase domain is present.
 42. A method of monitoring theformation of RAF/RAF or RAF/KSR kinase domain dimers to detect mutationsinhibiting dimerization or drug-like molecules interfering withdimerization, the method comprising: a) using either (i) a RAF kinasedomain or (ii) a KSR kinase domain at either of their N- or C-termini toa BRET donor or a BRET acceptor to produce donor labeled and acceptorlabeled fusion proteins; b) expressing the fusion proteins to identifycombinations that provide specific BRET signals; c) introducing dimerinterface mutations into either of the labeled fusion proteins; d)expressing the labeled mutated fusion proteins with WT RAF or KSR kinasedomains; e) measuring the BRET signals, a loss or significant reductionof the BRET signal using dimer interface mutations as opposed tomutations remote from the interface, being an indication that a specificBRET signal which depends on the RAF/RAF or RAF/KSR dimerizationinterface has been obtained.
 43. The method, according to claim 42, inwhich the BRET donor is renilla luciferase variant II or rlucII.
 44. Themethod, according to claim 42, in which the BRET acceptor is GFP10. 45.The method, according to claim 42, in which the acceptor label is YellowFluorescent Protein (YFP).
 46. The method, according to claim 42, inwhich the donor labeled fusion protein comprises a sequence selectedfrom the group consisting of: SEQ ID NO. 24, SEQ ID NO. 34, SEQ ID NO.42 and SEQ ID NO.
 48. 47. The method, according to claim 42, in whichthe acceptor labeled fusion protein comprises a sequence selected fromthe group consisting of: SEQ ID NO. 22, SEQ ID NO. 30, SEQ ID NO. 40 andSEQ ID NO.
 54. 48. The method, according to claim 42, in which the donorlabeled mutated fusion proteins comprise sequences SEQ ID NO. 36 and SEQID NO.
 50. 49. The method, according to claim 42, in which the acceptorlabeled mutated fusion proteins comprises a sequence of SEQ ID NO. 32.50. A method of identifying a potential inhibitor of RAF/RAFhomodimerization, the method comprising. a) fusing a RAF kinase domainat either of its N- or C-termini to a BRET donor or a BRET acceptor toproduce donor labeled and acceptor labeled fusion proteins; b)expressing the fusion proteins to identify combinations that providespecific BRET signals; c) introducing dimer interface mutations intoeither of the labeled fusion proteins; d) expressing the labeled mutatedfusion proteins with WT RAF kinase domains; e) contacting the interfacewith the potential inhibitor; and f) measuring the BRET signals, a lossor significant reduction of the BRET signal for the wild-type RAF/RAFBRET pair being an indication that the inhibitor is specifically boundto the interface.
 51. A method of identifying a potential inhibitor ofRAF/RAF homodimerization, the method comprising: a) detectably labelingat least one of the dimerization interface residues to generate adetectably labeled RAF monomer; b) incubating the detectably labeled RAFmonomer with the potential inhibitor and a non-labeled RAF monomer; c)measuring a signal from the detectable label; d) contacting the RAFdimerization interface with the inhibitor to determine the ability ofthe potential inhibitor to inhibit RAF/RAF homodimerization.
 52. Themethod, according to claim 51, in which the interface residues includeH449, G450, R481, L487, F488, M489, Y538, A541 or K542.
 53. A method ofidentifying a potential inhibitor of RAF/RAF homodimerization, themethod comprising: a) fusing a RAF kinase domain at either of its N- orC-termini to a BRET donor or a BRET acceptor to produce donor labeledand acceptor labeled fusion proteins; b) expressing the fusion proteinsto identify combinations that provide specific BRET signals; c)introducing dimer interface mutations into either of the labeled fusionproteins; d) expressing the labeled mutated fusion proteins with WT RAFkinase domains; e) contacting the interface with the potentialinhibitor; and f) measuring the BRET signals, a loss or significantreduction of the BRET signal for the wild-type RAF/RAF BRET pair beingan indication that the inhibitor is specifically bound to the interface.54. A method of identifying compounds that bind to a RAF or a KSRdimerization interface, the method comprising: a) contacting theinterface with a probe to form a probe: interface complex, the probebeing displaceable by a test compound; b) measuring a signal from theprobe so as to establish a reference level; c) incubating theprobe:interface complex with the test compound; d) measuring the signalfrom the probe; e) comparing the signal from step d) with the referencelevel, a modulation of the signal being an indication that the testcompound binds to the BIR domain, wherein the probe is a compoundlabeled with a detectable label or an affinity label.
 55. A method ofidentifying a potential inhibitor of RAF/RAF homodimerization, themethod comprising: a) using the atomic coordinates of at least one ofthe interface residues to generate a three dimensional structure of aRAF dimerization interface; b) using the three-dimensional structure todesign or select the potential inhibitor; c) synthesizing the inhibitor;and d) contacting the RAF dimierization interface with the inhibitor todetermine the ability of the potential inhibitor to inhibit RAF/RAFhomodimerization.
 56. The method, according to claim 55, in which theinterface residues are H449, G450, R481, L487, F488, M489, Y538, A541 orK542.
 57. A method of identifying a potential inhibitor of RAF/KSRheterodimerization, the method comprising: a) using the atomiccoordinates of at least one of interface residues to generate a threedimensional structure of a KSR dimerization interface; b) using thethree-dimensional structure to design or select the potential inhibitor;c) synthesizing the inhibitor; and d) contacting the KSR dimerizationinterface with the inhibitor to determine the ability of the potentialinhibitor to inhibit RAF/KSR heterodimerization.
 58. The method,according to claim 57, in which the interface residues are H699, G700,R732, L738, F739, M740, Y790, A793 or R794. 59-61. (canceled)